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Aims: Deregulation of mTOR (mammalian target of rapamycin) signalling occurs in diabetes, which exacerbates injury following myocardial infarction (MI). We therefore investigated the infarct-limiting effect of chronic treatment with rapamycin (RAPA, mTOR inhibitor) in diabetic mice following myocardial ischaemia/reperfusion (I/R) injury and delineated the potential protective mechanism., Methods and Results: Adult male diabetic (db/db) or wild-type (WT) (C57) mice were treated with RAPA (0.25 mg/kg/day, intraperitoneal) or vehicle (5% DMSO) for 28 days. The hearts from treated mice were subjected to global I/R in Langendorff mode. Cardiomyocytes, isolated from treated mice, were subjected to simulated ischaemia/reoxygenation (SI/RO) to assess necrosis and apoptosis. Myocardial infarct size was increased in diabetic heart following I/R as compared to WT. Likewise, enhanced necrosis and apoptosis were observed in isolated cardiomyocytes of diabetic mice following SI/RO. Treatment with RAPA reduced infarct size as well as cardiomyocyte necrosis and apoptosis of diabetes and WT mice. RAPA increased STAT3 phosphorylation and miRNA-17/20a expression in diabetic hearts. In addition, RAPA restored AKT phosphorylation (target of mTORC2) but suppressed S6 phosphorylation (target of mTORC1) following I/R injury. RAPA-induced cardioprotection against I/R injury as well as the induction of miR-17/20a and AKT phosphorylation were abolished in cardiac-specific STAT3-deficient diabetic mice, without alteration of S6 phosphorylation. The infarct-limiting effect of RAPA was obliterated in cardiac-specific miRNA-17-92-deficient diabetic mice. The post-I/R restoration of phosphorylation of STAT3 and AKT with RAPA were also abolished in miRNA-17-92-deficient diabetic mice. Additionally, RAPA suppressed the pro-apoptotic prolyl hydroxylase (Egln3/PHD3), a target of miRNA-17/20a in diabetic hearts, which was abrogated in miRNA-17-92-deficient diabetic mice., Conclusion: Induction of STAT3-miRNA-17-92 signalling axis plays a critical role in attenuating MI in RAPA-treated diabetic mice. Our study indicates that chronic treatment with RAPA might be a promising pharmacological intervention for attenuating MI and improving prognosis in diabetic patients., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Aims: Hydrogen sulfide (H 2 S) protects against ischemic and inflammatory injury following myocardial ischemia via induction of microRNA (miR)-21. We sought to determine whether H 2 S attenuates ischemic heart failure with reduced ejection fraction (HFrEF) and interrogate the role of cofilin-2, a target of miR-21, in this protective process., Methods and Results: Adult male mice underwent myocardial infarction (MI) by coronary artery ligation after baseline echocardiography. Following MI, mice were treated with Na 2 S (100 μg/kg/day; intraperitoneal [IP]) or saline up to 28 days. End-diastolic pressure, measured by Millar catheter, was significantly increased ( P < .05 vs sham) at 3 days post-MI in the saline group, which was attenuated with Na 2 S. Left ventricular (LV) fractional shortening decreased significantly at 28 days post-MI in the saline group but was preserved with Na 2 S and LV infarct scar size was smaller in Na 2 S group as compared to control. Apoptotic signaling, measured by Bcl-2/Bax ratio, was significantly increased in the saline group but was mitigated with Na 2 S. Survival rate was 2-fold higher in Na 2 S group compared to saline control ( P < .05). Proteomic analysis and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (TOF)/TOF tandem mass spectrometry identified significant changes in proapoptotic cofilin-2 expression, a specific target of miR-21, between saline- and sodium sulfide -treated mice at 28 days post-MI. Western blot analysis confirmed a significant increase in cofilin-2 after MI, which was suppressed with Na 2 S treatment. Chronic Na 2 S treatment also attenuated inflammasome formation and activation leading to reduction of maladaptive signaling., Conclusion: Na 2 S treatment after MI preserves LV function and improves survival through attenuation of inflammasome-mediated adverse remodeling. We propose H 2 S donors as promising therapeutic tools for ischemic HFrEF.

Background Human relaxin-2 is a peptide hormone capable of pleiotropic effects in several organ systems. Its recombinant formulation (serelaxin) has been demonstrated to reduce infarct size and prevent excessive scar formation in animal models of cardiac ischemia-reperfusion injury. B7-33, a synthetically designed peptide analogous to B-chain of relaxin-2, invokes signaling at relaxin family peptide receptor 1 (cognate receptor for relaxin-2) by preferentially phosphorylating the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2. We sought to investigate the effects of B7-33 treatment post ischemia-reperfusion injury in mice. Methods and Results Adult male CD1 mice were subjected to ischemia-reperfusion via ligation of left anterior descending artery for 30 minutes, followed by 24 hours or 7 days of reperfusion. Echocardiography was performed to assess cardiac function, and cardiac tissue was stained to determine infarct size at 24 hours. B7-33 significantly reduced infarct size (21.99% versus 45.32%; P =0.02) and preserved fractional shortening (29% versus 23%; P =0.02) compared with vehicle. The difference in fractional shortening further increased at 7 days post myocardial infarction (29% versus 20% for B7-33 and vehicle groups, respectively). In vitro , primary cardiomyocytes were isolated from adult hearts and subjected to simulated ischemia-reperfusion injury (simulated ischemia reoxygenation). B7-33 (50 and 100 nmol/L) improved cell survival and reduced the expression of GRP78 (glucose regulated protein), an endoplasmic reticulum stress marker. Subsequently, B7-33 (100 nmol/L) reduced tunicamycin (2.5 μg/mL) induced upregulation of GRP78 in an extracellular signal-regulated kinase 1/2-dependent manner. Conclusions B7-33 confers acute cardioprotection and limits myocardial infarction-related adverse remodeling in mice by attenuating cardiomyocyte death and endoplasmic reticulum stress as well as preserving cardiac function.

Background: Sacubitril/valsartan (SAC/VAL) is approved by the U.S. Food and Drug Administration for heart failure with reduced ejection fraction (HFrEF)., Objectives: This study investigated the effects of SAC/VAL on acute myocardial infarction (MI) and cardiac remodeling in a translational rabbit model of MI., Methods: New Zealand White rabbits were sedated and underwent conscious MI (45-min ischemia) by balloon inflation (previously implanted surgically) followed by 72 h (acute protocol) or 10 weeks (chronic protocols) of reperfusion. "Infarct-sparing" protocol: SAC/VAL, VAL, or placebo were randomly allocated and administered at reperfusion. "HFrEF-treatment" protocol: rabbits were randomized, and treatment commenced after echocardiography-confirmed left ventricular ejection fraction (LVEF) ≤40%. "HFrEF-prevention" protocol: treatment started at reperfusion and continued daily throughout the study., Results: Compared with placebo, SAC/VAL and VAL significantly reduced infarct size (TTC staining) and plasma troponin levels; however, only SAC/VAL preserved LVEF at 72 h post-MI. In the HFrEF-treatment protocol, LVEF improvement was observed with SAC/VAL compared with both placebo and VAL starting 2 weeks post-treatment, a benefit that persisted throughout study duration. In the HFrEF-prevention protocol, SAC/VAL and VAL attenuated the decline in LVEF post-MI, although SAC/VAL offered better functional protection. The functional improvement observed in both treatment protocols was paralleled by significant reduction in left ventricular (LV) scar size (Picrosirius red staining) in the SAC/VAL groups., Conclusions: Reperfusion therapy with SAC/VAL or VAL offers robust acute infarct-sparing benefits; however, SAC/VAL treatment offered superior short-term and long-term benefits in preventing MI-induced LV dysfunction compared with VAL. SAC/VAL also significantly attenuated LV scar size following MI compared with placebo, whereas VAL did not reach statistical significance in scar reduction., (Copyright © 2018 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: The area of myocardial infarction continues to expand for hours after reperfusion. The injured but viable myocardium may be salvaged if the signals leading to cell death are interrupted. Activation of the caspase-1 inflammasome in the heart shortly after ischemia-reperfusion contributes to the final infarct size. Plasma-derived α-1 anti-trypsin (AAT) has shown to inhibit inflammasome formation in vitro and in vivo. To explore the potential translational clinical value of AAT as a therapeutic, we conducted a series of preclinical experiments designed to simulate clinically relevant scenarios., Methods: Adult male CD1 mice were used. The left anterior descending coronary artery was ligated for 30 or 75 minutes followed by reperfusion, to explore different severity of ischemic injury. Plasma-derived AAT (Prolastin C) was administered intraperitoneally after reperfusion, without pretreatment, exploring 3 different doses (60, 120, and 180 mg/kg). In a subgroup of mice, we administered Prolastin C with a delay of 30 minutes after reperfusion to simulate the clinical context of delayed administration, and we also used a model of permanent coronary artery ligation without reperfusion. Finally, we tested whether a single dose at reperfusion was sufficient to maintain a benefit in the longer term (7 days). Infarct size was measured by 3 different and independent methodologies: pathology, plasma levels of troponin I, and wall motion abnormalities at echocardiography., Results: Prolastin C given at reperfusion after 30 minutes of ischemia provided a powerful reduction in infarct size (>50% reduction in all methodology used, all P < 0.01) without a clear dose-dependent response. Prolongation of ischemia to 75 minutes nor a delay in treatment by 30 minutes after reperfusion had any negative impact on Prolastin C effects. A single dose given at reperfusion was as effective as multiple daily doses. When given to the mouse without reperfusion, Prolastin C failed to reduce infarct size., Conclusions: Plasma-derived AAT (Prolastin C) given as an adjunct to reperfusion powerfully limits the final infarct size across a wide range of experiments in the mouse reproducing clinically relevant scenarios, such as variable duration of ischemia, delay in administration in the drug, and a large therapeutic index.

Aims: The preconditioning-like infarct-sparing and anti-inflammatory effects of the peptide hormone relaxin following ischemic injury have been studied in the heart. Whether reperfusion therapy with recombinant human relaxin-2, serelaxin, reduces myocardial infarct size and attenuates the subsequent NLRP3 inflammasome activation leading to further loss of functional myocardium following ischemia/reperfusion (I/R) injury is unknown., Methods and Results: After baseline echocardiography, adult male wild-type C57BL or eNOS knockout mice underwent myocardial infarction (MI) by coronary artery ligation for 30 min followed by 24 h reperfusion. Mice were treated with either serelaxin (10 µg/kg; sc) or saline 1 h prior to ischemia or 5 min before reperfusion. In both pre-treatment and reperfusion therapy arms, serelaxin improved survival at 24 h post MI in wild-type mice (79% and 82%) as compared with controls (46% and 50%, P = 0.01), whereas there was no difference in survival between serelaxin- and saline-treated eNOS knockout mice. Moreover, serelaxin significantly reduced infarct size (64% and 67% reduction, P < 0.05), measured with TTC staining, and preserved LV fractional shortening (FS) and end-systolic diameter (LVESD) in wild-type mice as compared with controls (P < 0.05). Interestingly, caspase-1 activity in the heart tissue, a measure of inflammasome formation, was markedly reduced in serelaxin-treated wild-type mice compared with controls at 24 h post-MI in both treatment modalities (P < 0.05). Genetic deletion of eNOS abolished the infarct-sparing and anti-inflammatory effects of serelaxin as well as functional preservation. Serelaxin plasma levels assessed at 5 min and 1 h after treatment, using ELISA, approximated physiologic relaxin levels during pregnancy in mice and parallels that in humans., Conclusion: Serelaxin attenuates myocardial I/R injury and the subsequent caspase-1 activation via eNOS-dependent mechanism., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Prompt coronary reperfusion is the gold standard for minimizing injury following acute myocardial infarction. Rapamycin, mammalian target of Rapamycin (mTOR) inhibitor, exerts preconditioning-like cardioprotective effects against ischemia/reperfusion (I/R) injury. We hypothesized that Rapamycin, given at the onset of reperfusion, reduces myocardial infarct size through modulation of mTOR complexes. Adult C57 male mice were subjected to 30 min of myocardial ischemia followed by reperfusion for 1 hour/24 hours. Rapamycin (0.25 mg/kg) or DMSO (7.5%) was injected intracardially at the onset of reperfusion. Post-I/R survival (87%) and cardiac function (fractional shortening, FS: 28.63 ± 3.01%) were improved in Rapamycin-treated mice compared to DMSO (survival: 63%, FS: 17.4 ± 2.6%). Rapamycin caused significant reduction in myocardial infarct size (IS: 26.2 ± 2.2%) and apoptosis (2.87 ± 0.64%) as compared to DMSO-treated mice (IS: 47.0 ± 2.3%; apoptosis: 7.39 ± 0.81%). Rapamycin induced phosphorylation of AKT S473 (target of mTORC2) but abolished ribosomal protein S6 phosphorylation (target of mTORC1) after I/R. Rapamycin induced phosphorylation of ERK1/2 but inhibited p38 phosphorylation. Infarct-limiting effect of Rapamycin was abolished with ERK inhibitor, PD98059. Rapamycin also attenuated Bax and increased Bcl-2/Bax ratio. These results suggest that reperfusion therapy with Rapamycin protects the heart against I/R injury by selective activation of mTORC2 and ERK with concurrent inhibition of mTORC1 and p38., Competing Interests: On behalf of all authors, the corresponding author states that there is no conflict of interests.

Ingestion of high dietary nitrate in the form of beetroot juice (BRJ) has been shown to exert antihypertensive effects in humans through increasing cyclic guanosine monophosphate (cGMP) levels. Since enhanced cGMP protects against myocardial ischemia-reperfusion (I/R) injury through upregulation of hydrogen sulfide (H2S), we tested the hypothesis that BRJ protects against I/R injury via H2S. Adult male CD-1 mice received either regular drinking water or those dissolved with BRJ powder (10 g/L, containing ∼ 0.7 mM nitrate). Seven days later, the hearts were explanted for molecular analyses. Subsets of mice were subjected to I/R injury by occlusion of the left coronary artery for 30 min and reperfusion for 24 h. A specific inhibitor of H2S producing enzyme--cystathionine-γ-lyase (CSE), DL-propargylglycine (PAG, 50 mg/kg) was given i.p. 30 min before ischemia. Myocardial infarct size was significantly reduced in BRJ-fed mice (15.8 ± 3.2%) versus controls (46.5 ± 3.5%, mean ± standard error [SE], n = 6/group, P < .05). PAG completely blocked the infarct-limiting effect of BRJ. Moreover, BRJ significantly preserved ventricular function following I/R. Myocardial levels of H2S and its putative protein target--vascular endothelial growth factor receptor 2 (VEGFR2) were significantly increased by BRJ intake, whereas CSE mRNA and protein content did not change. Interestingly, the BRJ-induced cardioprotection was not associated with elevated blood nitrate-nitrite levels following I/R nor induction of cardiac peroxiredoxin 5, a mitochondrial antioxidant enzyme previously linked to nitrate-induced cardioprotection. We conclude that BRJ ingestion protects against post-I/R myocardial infarction and ventricular dysfunction possibly through CSE-mediated endogenous H2S generation. BRJ could be a promising natural and inexpensive nutraceutical supplement to reduce cardiac I/R injury in patients., (© 2014 by the Society for Experimental Biology and Medicine.)

Rationale: Despite 4 decades of intense effort and substantial financial investment, the cardioprotection field has failed to deliver a single drug that effectively reduces myocardial infarct size in patients. A major reason is insufficient rigor and reproducibility in preclinical studies., Objective: To develop a multicenter, randomized, controlled, clinical trial-like infrastructure to conduct rigorous and reproducible preclinical evaluation of cardioprotective therapies., Methods and Results: With support from the National Heart, Lung, and Blood Institute, we established the Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR), based on the principles of randomization, investigator blinding, a priori sample size determination and exclusion criteria, appropriate statistical analyses, and assessment of reproducibility. To validate CAESAR, we tested the ability of ischemic preconditioning to reduce infarct size in 3 species (at 2 sites/species): mice (n=22-25 per group), rabbits (n=11-12 per group), and pigs (n=13 per group). During this validation phase, (1) we established protocols that gave similar results between centers and confirmed that ischemic preconditioning significantly reduced infarct size in all species and (2) we successfully established a multicenter structure to support CAESAR's operations, including 2 surgical centers for each species, a Pathology Core (to assess infarct size), a Biomarker Core (to measure plasma cardiac troponin levels), and a Data Coordinating Center-all with the oversight of an external Protocol Review and Monitoring Committee., Conclusions: CAESAR is operational, generates reproducible results, can detect cardioprotection, and provides a mechanism for assessing potential infarct-sparing therapies with a level of rigor analogous to multicenter, randomized, controlled clinical trials. This is a revolutionary new approach to cardioprotection. Importantly, we provide state-of-the-art, detailed protocols ("CAESAR protocols") for measuring infarct size in mice, rabbits, and pigs in a manner that is rigorous, accurate, and reproducible., (© 2014 American Heart Association, Inc.)

Galectin-1 (Gal-1), an evolutionarily conserved β-galactoside-binding lectin, plays essential roles in the control of inflammation and neovascularization. Although identified as a major component of the contractile apparatus of cardiomyocytes, the potential role of Gal-1 in modulating heart pathophysiology is uncertain. Here, we aimed to characterize Gal-1 expression and function in the infarcted heart. Expression of Gal-1 was substantially increased in the mouse heart 7 days after acute myocardial infarction (AMI) and in hearts from patients with end-stage chronic heart failure. This lectin was localized mainly in cardiomyocytes and inflammatory infiltrates in peri-infarct areas, but not in remote areas. Both simulated hypoxia and proinflammatory cytokines selectively up-regulated Gal-1 expression in mouse cardiomyocytes, whereas anti-inflammatory cytokines inhibited expression of this lectin or had no considerable effect. Compared with their wild-type counterpart, Gal-1-deficient (Lgals1(-/-)) mice showed enhanced cardiac inflammation, characterized by increased numbers of macrophages, natural killer cells, and total T cells, but reduced frequency of regulatory T cells, leading to impaired cardiac function at baseline and impaired ventricular remodeling 7 days after nonreperfused AMI. Treatment of mice with recombinant Gal-1 attenuated cardiac damage in reperfused AMI. Taken together, our results indicate a protective role for Gal-1 in normal cardiac homeostasis and postinfarction remodeling by preventing cardiac inflammation. Thus, Gal-1 treatment represents a potential novel strategy to attenuate heart failure in AMI., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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 (H(2)S). 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 H(2)S-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 H(2)S levels, which was abrogated by KT. Cinaciguat is a novel molecule to induce H(2)S generation and a powerful protection against I/R injury in heart.

The rationale of this article is enhancing the therapeutic potential of stem cells in ischemic microenvironments by novel preconditioning strategies is critical for improving cellular therapy. We tested the hypothesis that inhibition of phosphodiesterase-5 (PDE-5) with sildenafil (Viagra) or knockdown with a silencing vector in adipose-derived stem cells (ASCs) would improve their survival and enhance cardiac function following myocardial implantation in vivo. ASCs were treated with sildenafil or PDE-5 silencing vector short hairpin RNA (shRNA(PDE-5)) and subjected to simulated ischemia/reoxygenation in vitro. Both sildenafil and shRNA(PDE-5) significantly improved viability, decreased necrosis, apoptosis, and enhanced the release of growth factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), and insulin-like growth factor. Inhibition of protein kinase G reversed these effects. To show the beneficial effect of preconditioned ASCs in vivo, adult male CD-1 mice underwent myocardial infarction. Preconditioned ASCs (4 × 10(5)) were directly injected intramyocardially. Preconditioned ASC-treated hearts showed consistently superior cardiac function when compared with nonpreconditioned ASCs after 4 weeks of treatment. This was associated with significantly reduced fibrosis, increased vascular density, and decreased resident myocyte apoptosis when compared with mice receiving nonpreconditioned ASCs. VEGF, b-FGF, and Angiopoietin-1 were also significantly elevated 4 weeks after cell therapy with preconditioned ASCs. We conclude that preconditioning by inhibition of PDE-5 can be a powerful novel approach to improve stem cell therapy following myocardial infarction., (Copyright © 2011 AlphaMed Press.)

Background: Insulin resistance impairs nitric oxide (NO) bioavailability and obesity promotes a state of chronic inflammation and damages the vascular endothelium. Phosphodiesterase-5 inhibitors restore NO signaling and may reduce circulating inflammatory markers, and improve metabolic parameters through a number of mechanisms. We hypothesized that daily administration of the PDE-5 inhibitor, tadalafil (TAD) will attenuate inflammation, improve fasting plasma glucose and triglyceride levels, body weight, and reduce infarct size after ischemia/reperfusion injury in obese, diabetic mice., Methods: Twenty leptin receptor null (db/db) mice underwent treatment with TAD (1 mg/Kg) or 10% DMSO for 28 days. Body weight and fasting plasma glucose levels were determined weekly. Upon completion, hearts were isolated and subjected to 30 min global ischemia followed by 60 min reperfusion in a Langendorff model. Plasma samples were taken for cytokine analysis and fasting triglyceride levels. Infarct size was measured using computer morphometry of tetrazolium stained sections. Additionally, ventricular cardiomyocytes were isolated and subjected to 40 min of simulated ischemia and reoxygenation. Necrosis was determined using trypan blue exclusion and LDH release assay and apoptosis was assessed by TUNEL assay after 1 h or 18 h of reoxygenation, respectively., Results: Treatment with TAD caused a reduction in infarct size in the diabetic heart (23.2 ± 1.5 vs. 47.8 ± 3.7%, p<0.01, n = 6/group), reduced fasting glucose levels (292 ± 31.8 vs. 511 ± 19.3 mg/dL, p<0.001) and fasting triglycerides (43.3 ± 21 vs. 129.7 ± 29 mg/dL, p<0.05) as compared to DMSO, however body weight was not significantly reduced. Circulating tumor necrosis factor-α and interleukin-1β were reduced after treatment compared to control (257 ± 16.51 vs. 402.3 ± 17.26 and 150.8 ± 12.55 vs. 264 ± 31.85 pg/mL, respectively; P<0.001) Isolated cardiomyocytes from TAD-treated mice showed reduced apoptosis and necrosis., Conclusion: We have provided the first evidence that TAD therapy ameliorates circulating inflammatory cytokines and chemokines in a diabetic animal model while improving fasting glucose levels and reducing infarct size following ischemia-reperfusion injury in the heart.

Acute myocardial infarction (AMI) initiates an intense inflammatory response that promotes cardiac dysfunction, cell death, and ventricular remodeling. The molecular events underlying this inflammatory response, however, are incompletely understood. In experimental models of sterile inflammation, ATP released from dying cells triggers, through activation of the purinergic P2X7 receptor, the formation of the inflammasome, a multiprotein complex necessary for caspase-1 activation and amplification of the inflammatory response. Here we describe the presence of the inflammasome in the heart in an experimental mouse model of AMI as evidenced by increased caspase-1 activity and cytoplasmic aggregates of the three components of the inflammasome--apoptosis speck-like protein containing a caspase-recruitment domain (ASC), cryopyrin, and caspase-1, localized to the granulation tissue and cardiomyocytes bordering the infarct. Cultured adult murine cardiomyocytes also showed the inducible formation of the inflammasome associated with increased cell death. P2X7 and cryopyrin inhibition (using silencing RNA or a pharmacologic inhibitor) prevented the formation of the inflammasome and limited infarct size and cardiac enlargement after AMI. The formation of the inflammasome in the mouse heart during AMI causes additional loss of functional myocardium, leading to heart failure. Modulation of the inflammasome may therefore represent a unique therapeutic strategy to limit cell death and prevent heart failure after AMI.

Background: Cardiac remodelling after AMI is characterized by molecular and cellular mechanisms involving both the ischemic and non-ischemic myocardium. The extent of right ventricular (RV) dilatation and dysfunction and its relation to pulmonary hypertension (PH) following AMI are unknown. The aim of the current study was to evaluate changes in dimensions and function of the RV following acute myocardial infarction (AMI) involving the left ventricle (LV)., Methods: We assessed changes in RV dimensions and function 1 week following experimental AMI involving the LV free wall in 10 mice and assessed for LV and RV dimensions and function and for the presence and degree of PH., Results: RV fractional area change and tricuspidal annular plane systolic excursion significantly declined by 33% (P = 0.021) and 28% (P = 0.001) respectively. Right ventricular systolic pressure measured invasively in the mouse was within the normal values and unchanged following AMI., Conclusion: AMI involving the LV and sparing the RV induces a significant acute decline in RV systolic function in the absence of pulmonary hypertension in the mouse indicating that RV dysfunction developed independent of changes in RV afterload.

Background: Healing after acute myocardial infarction (AMI) is characterized by an intense inflammatory response and increased Interleukin-1 (IL-1) tissue activity. Genetically engineered mice lacking the IL-1 receptor (IL-1R1-/-, not responsive to IL-1) or the IL-1 receptor antagonist (IL-1Ra, enhanced response to IL-1) have an altered IL-1/IL-1Ra balance that we hypothesize modulates infarct healing and cardiac remodeling after AMI., Methods: IL-1R1-/- and IL-1Ra-/- male mice and their correspondent wild-types (WT) were subjected to permanent coronary artery ligation or sham surgery. Infarct size (trichrome scar size), apoptotic cell death (TUNEL) and left ventricular (LV) dimensions and function (echocardiography) were measured prior to and 7 days after surgery., Results: When compared with the corresponding WT, IL-1R1-/- mice had significantly smaller infarcts (-25%), less cardiomyocyte apoptosis (-50%), and reduced LV enlargement (LV end-diastolic diameter increase [LVEDD], -20%) and dysfunction (LV ejection fraction [LVEF] decrease, -50%), whereas IL-1Ra-/- mice had significantly larger infarcts (+75%), more apoptosis (5-fold increase), and more severe LV enlargement (LVEDD increase,+30%) and dysfunction (LVEF decrease, +70%)(all P values <0.05)., Conclusions: An imbalance in IL-1/IL-1Ra signaling at the IL-1R1 level modulates the severity of cardiac remodeling after AMI in the mouse, with reduced IL-1R1 signaling providing protection and unopposed IL-1R1 signaling providing harm.

Acute myocardial infarction (AMI) initiates an intense inflammatory response in which interleukin-1 (IL-1) plays a central role. The IL-1 receptor antagonist is a naturally occurring antagonist, and anakinra is the recombinant form used to treat inflammatory diseases. The aim of the present pilot study was to test the safety and effects of IL-1 blockade with anakinra on left ventricular (LV) remodeling after AMI. Ten patients with ST-segment elevation AMI were randomized to either anakinra 100 mg/day subcutaneously for 14 days or placebo in a double-blind fashion. Two cardiac magnetic resonance (CMR) imaging and echocardiographic studies were performed during a 10- to 14-week period. The primary end point was the difference in the interval change in the LV end-systolic volume index (LVESVi) between the 2 groups on CMR imaging. The secondary end points included differences in the interval changes in the LV end-diastolic volume index, and C-reactive protein levels. A +2.0 ml/m(2) median increase (interquartile range +1.0, +11.5) in the LVESVi on CMR imaging was seen in the placebo group and a -3.2 ml/m(2) median decrease (interquartile range -4.5, -1.6) was seen in the anakinra group (p = 0.033). The median difference was 5.2 ml/m(2). On echocardiography, the median difference in the LVESVi change was 13.4 ml/m(2) (p = 0.006). Similar differences were observed in the LV end-diastolic volume index on CMR imaging (7.6 ml/m(2), p = 0.033) and echocardiography (9.4 ml/m(2), p = 0.008). The change in C-reactive protein levels between admission and 72 hours after admission correlated with the change in the LVESVi (R = +0.71, p = 0.022). In conclusion, in the present pilot study of patients with ST-segment elevation AMI, IL-1 blockade with anakinra was safe and favorably affected by LV remodeling. If confirmed in larger trials, IL-1 blockade might represent a novel therapeutic strategy to prevent heart failure after AMI., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Myeloid differentiation factor 88 (MyD88) is an endogenous adaptor protein that coordinates the inflammatory response to agonists of the Toll-like receptor and interleukin-1 receptor families. This particular response is activated following myocardial ischemia and infarction and may represent a viable target for pharmacologic inhibition. The current study tested MyD88 inhibitors in a murine model of nonreperfused acute myocardial infarction (AMI)., Methods: AMI was induced by permanent ligation of the left coronary artery. Adult, male, Imprinting Control Region mice were randomized to daily injections with 1 of 2 MyD88 pharmacologic inhibitors (ST2825 25 mg/kg or IMG2005 1 mg/kg), saline, or pretreatment with MyD88-targeted silencing small interfering RNA (siRNA) or scrambled nontargeted siRNA (n = 6 for each group). Echocardiography was performed at baseline and 7 days after surgery to evaluate pathologic cardiac enlargement., Results: Pharmacologic inhibition of MyD88 with ST2825 or IMG2005) and MyD88-targeted siRNA protected against left ventricular (LV) dilatation (reduced LV end-systolic and LV end-diastolic diameter) and hypertrophy. This protection occurred despite no measurable reduction in infarct size., Conclusions: Pharmacologic MyD88 inhibition protects against pathologic LV remodeling without altering infarct scar formation. MyD88 may be a viable target for pharmacologic inhibition in AMI.

Background: Interleukin-1 (IL-1) is an inflammatory cytokine that responds as an acute phase reactant during acute myocardial infarction. Conflicting data describe the role of anti-IL-1 interventions to reduce cardiac remodeling after AMI. IL-1 Trap is a modified recombinant fusion protein that binds circulating IL-1. Our study evaluated the effects of murine IL-1 Trap on cardiac remodeling after AMI resulting from permanent surgical coronary artery ligation., Methods: Mice received treatment with intraperitoneal injection of murine IL-1 Trap (1 mg/kg [n = 5], 5 mg/kg [n = 5], or 30 mg/kg [n = 5]) or NaCl 0.9% (saline; n = 10) every 48 hours after surgery. Transthoracic echocardiography was performed at baseline and 7 days after surgery. Inhibition of IL-1 signaling was determined by measurement of IL-6 plasma levels (enzyme-linked immunosorbent assay) after IL-1b injection. Apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) was measured in murine heart samples and in a primary culture of murine cardiomyocytes., Results: Mice treated with 5 mg/kg or 30 mg/kg IL-1 Trap had more favorable cardiac remodeling and echocardiographic assessment of infarct size at 7 days compared with saline (P < 0.05 for each comparison). Treatment with IL-1 Trap also reduced apoptosis and IL-6 levels compared with saline treatment., Conclusions: IL-1 Trap ameliorates cardiac remodeling and reduces cardiomyocyte apoptosis after experimental acute myocardial infarction in the mouse.

Purpose: Myocardial ischemia induces cyclooxygenase 2 (COX-2) expression. We evaluated the effects of parecoxib, a COX-2 inhibitor, in 2 different mouse models of myocardial ischemia: permanent left coronary artery ligation (PI) and transient ligation (30 minutes ischemia) followed by reperfusion (I/R)., Methods: Forty adult male Institute of Cancer Research mice underwent PI (n = 24) or I/R (n = 16), followed by randomization to parecoxib (0.75 mg/kg intraperitoneal daily) or normal saline for 7 days., Results: Parecoxib significantly reduced apoptosis [0.8% vs. 3.4% (saline), P < 0.001] and 7-day mortality [0% vs. 57% (saline), P = 0.040] in the PI group but showed no benefit in the I/R group. Parecoxib-treated mice also exhibited greater fractional shortening in the PI group [22% vs. 14% (saline), P = 0.045) but not in the I/R group. Parecoxib did not affect infarct size in either group., Conclusions: COX-2 may play a pivotal role in mediating apoptosis in the ischemic peri-infarct myocardium that is not reperfused after infarct.

Sildenafil, a selective inhibitor of phosphodiesterase type 5, induces powerful protection against myocardial ischemia-reperfusion injury through activation of cGMP-dependent protein kinase (PKG). We further hypothesized that PKG-dependent activation of survival kinase ERK may play a causative role in sildenafil-induced cardioprotection via induction of endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) and Bcl-2. Our results show that acute intracoronary infusion of sildenafil in Langendorff isolated mouse hearts before global ischemia-reperfusion significantly reduced myocardial infarct size (from 29.4 +/- 2.4% to 15.9 +/- 3.0%; P < 0.05). Cotreatment with ERK inhibitor PD98059 abrogated sildenafil-induced protection (31.8 +/- 4.4%). To further evaluate the role of ERK in delayed cardioprotection, mice were treated with sildenafil (ip) 24 h before global ischemia-reperfusion. PD98059 was administered (ip) 30 min before sildenafil treatment. Infarct size was reduced from 27.6 +/- 3.3% in controls to 7.1 +/- 1.5% in sildenafil-treated mice (P < 0.05). The delayed protective effect of sildenafil was also abolished by PD98059 (22.5 +/- 2.3%). Western blots revealed that sildenafil significantly increased phosphorylation of ERK1/2 and GSK-3beta and induced iNOS, eNOS, Bcl-2, and PKG activity in the heart 24 h after treatment. PD98059 inhibited the enhanced expression of iNOS, eNOS, and Bcl-2 and the phosphorylation of GSK-3beta. PD98059 had no effect on the sildenafil-induced activation of PKG. We conclude that these studies provide first direct evidence that PKG-dependent ERK phosphorylation is indispensable for the induction of eNOS/iNOS and Bcl-2 and the resulting cardioprotection by sildenafil.

Purpose: Interleukin-1 (IL-1) receptor antagonist (Ra) is a naturally occurring IL-1 blocker with a cardioprotective effect during acute myocardial infarction (AMI). Anakinra, recombinant-human IL-1Ra, has been used to prevent heart failure in a mouse model of AMI. The aim of this study was to determine the optimal therapeutic regimen for anakinra in AMI., Methods: We performed dose-response experiments comparing anakinra 1 mg/kg with 100 mg/kg doses, and duration-response experiments comparing 1-week to 2-week treatment. Echocardiography was used to assess cardiac remodeling and systolic function. Histopathology was used to detect apoptotic cardiomyocytes., Results: A higher dose of anakinra was not associated with additional improvement in cardiac remodeling or function. The 2-week anakinra treatment had sustained and more favorable remodeling and systolic function compared to 1-week treatment with significantly smaller left ventricular end-systolic diameter and greater fractional shortening 4 weeks after AMI., Conclusion: Anakinra inhibits apoptosis and ameliorates cardiac remodeling up to 4 weeks after infarction. A 2-week regimen is superior to a 1-week regimen, whereas a higher dose did not provide any further benefit over standard doses.

MicroRNAs (miRNAs) are noncoding RNAs of 18 to 24 nucleotides that are involved in posttranscriptional regulation of protein expression. Their role in ischemic preconditioning (IPC) is currently unknown. We hypothesized that miRNAs induced after IPC in the heart may create a preconditioned phenotype through upregulating proteins including endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) and heat shock protein (HSP)70, which are implicated in the late-phase protection of IPC. miRNAs were extracted from hearts of ICR mice following IPC. The purified miRNAs were injected in vivo into the left ventricular wall of mice, and, 48 hours later, the hearts were subjected to regional ischemia/reperfusion injury by left anterior descending artery ligation for 30 minutes followed by reperfusion for 24 hour. IPC caused no changes in miRNA-23b and miRNA-483 whereas miRNA-1, miRNA-21and miRNA-24 were significantly increased. The IPC-miRNA treatment caused an increase in eNOS mRNA and protein, whereas iNOS was not changed. HSF-1 (heat shock transcription factor 1) and HSP70 were also increased with IPC-miRNA treatment versus control. Moreover, injection of IPC-miRNA protected the hearts against ischemia/reperfusion injury, as shown by a reduction of infarct size as compared with saline or non-IPC miRNA-treated control. We conclude that IPC-induced miRNAs trigger cardioprotection similar to the delayed phase of IPC, possibly through upregulating eNOS, HSP70, and the HSP70 transcription factor HSF-1.

Cardiac remodeling after acute myocardial infarction (AMI) is characterized by molecular and cellular mechanisms involving both the left (LV) and right ventricular (RV) walls. Cardiomyoycte apoptosis in the peri-infarct and remote LV myocardium has a central role in cardiac remodeling. Whether apoptosis also occurs in the right ventricle of patients with ischemic heart disease has not been investigated. The aim of the present study was to investigate the presence of cardiomyocyte apoptosis in the right ventricle in patients with AMI. We assessed the number of apoptotic cardiomyocytes using multiple samplings in the LV and RV walls of 12 patients selected at autopsy who died 4 to 42 days after AMI. Five patients without cardiac disease were also selected at autopsy as controls. Apoptotic rates were calculated from the number of cardiomyocytes showing double positive staining for in situ end-labeling of DNA fragmentation (TUNEL) and for activated caspase-3. Potentially false-positive results (DNA synthesis and RNA splicing) were excluded from cell counts. The apoptotic rate in the right ventricle in patients with AMI was significantly higher than in control hearts (median 0.8%, interquartile range 0.3 to 1.0 vs median 0.01%, interquartile range 0.01 to 0.03, p <0.001). RV apoptosis significantly correlated with such parameters of global adverse remodeling as cardiac diameter to LV free wall thickness (R = +0.57, p = 0.050). RV apoptosis was significantly higher in five cases (42%) with infarct involving the ventricular septum and an adjacent small area of the RV walls (median 1.0%, interquartile range 0.8 to 2.2 vs median 0.5%, interquartile range 0.2 to 1.0, p = 0.048, p <0.001 vs controls). The association between apoptotic rate in the right ventricle and cardiac remodeling was apparent even after exclusion of cases with RV AMI involvement (R = +0.82, p = 0.023 for diameter to LV wall thickness ratio and R = -0.91, p = 0.002 for RV free wall thickness). In conclusion, patients with cardiac remodeling after AMI had a significant increase in RV apoptosis even when ischemic involvement of the RV wall was not apparent.

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 [n=6], 10 mg/kg [n=6], and 100 mg/kg [n=10]); 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 (P=0.013). 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 (P=0.015). Treatment with anakinra was associated with a significant reduction in cardiomyocyte apoptosis in both the immediate and delayed treatment groups (3.1+/-0.2% versus 0.5+/-0.3% [P<0.001] and 4.2+/-0.4% versus 1.1+/-0.2% [P<0.001], 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.

Objective: Selective cyclo-oxygenase-2 (COX-2) inhibitors have been shown to preserve hemodynamic performance in experimental models of acute myocardial infarction (AMI) in rodents. The impact of COX-2 inhibition on apoptosis, vascular density, and postinfarction remodeling has not yet been fully characterized. The aim of the present study was to evaluate the effects of parecoxib, a selective COX-2 inhibitor, in an experimental AMI model in the rat., Methods: Twenty-four male Wistar rats (10 weeks of age, weighing 350-500 g) underwent surgical left coronary artery ligation. Four animals died within 24 hours. Starting on day 2, 10 rats received parecoxib (0.75 mg/kg intraperitoneal) daily for 5 days and the remaining 10 received NaCl-0.9%. Animals underwent transthoracic echocardiography before surgery and 7 days later for the measurement of end-diastolic and end-systolic diameter and wall thickness; thereafter, animals were sacrificed and histological analysis was performed to evaluate cardiomyocyte apoptosis and small arteriolar density. Data are expressed as mean and standard error., Results: Three saline-treated (30%) and zero parecoxib-treated animals died before day 7. Compared with saline-treated animals, rats treated with parecoxib had a smaller end-diastolic diameter (6.3 +/- 0.1 vs. 7.0 +/- 0.1 mm, P = 0.018) and end-systolic diameter (2.7 +/- 0.1 vs. 3.9 +/- 0.1 mm, P = 0.027), and had a greater fractional shortening (57 +/- 1 vs. 45 +/- 2%, P = 0.050). Systolic thickness in the anterior (infarct) wall was also significantly greater in the parecoxib-treated animals (3.2 +/- 0.1 vs. 2.7 +/- 0.1 mm, P = 0.008), while the posterior wall was not significantly affected (P = 0.08). Aneurysmal dilatation of the left ventricle was more frequent in saline-treated versus parecoxib-treated animals (43 vs. 0%, P = 0.025). Parecoxib treatment was associated with lower apoptotic rates (1.0 +/- 0.2 vs. 4.0 +/- 0.4%, P < 0.001) and preservation of arteriolar density (20 +/- 5 vs. 8 +/- 2 mm/mm3, P = 0.018) in the peri-infarct area, without differences in circulating interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma levels., Conclusion: Administration of parecoxib significantly ameliorates the remodeling process after AMI, possibly through prevention of apoptosis and preservation of myocardial vascularity. These findings aid in the understanding of the role of COX-2 in ischemic damage and remodeling.

Objectives: The aim of the study was to analyze the molecular mechanisms activated during postinfarction remodeling in human hearts., Background: The molecular mechanisms of initial response to ischemic insult in the heart and the pathways involved in compensation and remodeling are still largely unknown., Methods: Up-regulation or down-regulation of gene expression in the human viable peri-infarct (vs. remote) myocardial region was investigated by complementary deoxyribonucleic acid array technology and confirmed at a single-gene/protein level with reverse transcriptase polymerase chain reaction and immunohistochemistry. An in vitro model of cardiomyocyte hypoxia in HL1 cells was used to validate anti-apoptotic effects of the candidate gene/protein and to assess the associated downstream cascade. Finally, a mouse model of myocardial infarction was used to test the in vivo effects of exogenous transfection with the candidate gene/protein., Results: Protein disulfide isomerase (PDI), a member of the unfolded protein response, is 3-fold up-regulated in the viable peri-infarct myocardial region, and in a postmortem model, its expression is significantly inversely correlated with apoptotic rate and with presence of heart failure (HF) and biventricular dilatation. Induced PDI expression in HL1 cells conferred protection from hypoxia-induced apoptosis. Adenoviral-mediated PDI gene transfer to the mouse heart resulted in 2.5-fold smaller infarct size, significantly reduced cardiomyocyte apoptosis in the peri-infarct region, and smaller left ventricular end-diastolic diameter versus mice treated with a transgene-null adenoviral vector., Conclusions: These results suggest that PDI promotes survival after ischemic damage and that zinc-superoxide dismutase is one of the PDI molecular targets. Pharmacological modulation of this pathway might prove useful for future prevention and treatment of HF.

Phosphodiesterase-5 (PDE-5) inhibitors including sildenafil and vardenafil induce powerful preconditioning-like cardioprotective effect against ischemia/reperfusion injury through opening of mitochondrial K(ATP) 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 K(ATP) 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; i.v.) infused for 65 min starting 5 min before reperfusion; 3-Sildenafil+5-hydroxydecanoate (5-HD, blocker of mitochondrial K(ATP) 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 microg 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 K(ATP) channel opening.

cGMP and opening of mitochondrial K(ATP) 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 K(ATP) 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 K(ATP) 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 K(ATP) 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.