ABSTRACT
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-ß, 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.
Subject(s)
Diabetes Mellitus , Myocardial Reperfusion Injury , Animals , Rabbits , Sirolimus/pharmacology , Sirolimus/therapeutic use , Ventricular Remodeling , Myocytes, Cardiac/metabolism , TOR Serine-Threonine Kinases/metabolism , Myocardial Reperfusion Injury/metabolism , Diabetes Mellitus/pathology , Inflammation/drug therapy , Inflammation/pathology , Ischemia/pathology , Fibrosis , Mammals/metabolismABSTRACT
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.
Subject(s)
Cardiovascular Agents/pharmacology , Drug Evaluation, Preclinical , Ischemic Preconditioning, Myocardial/methods , Myocardial Infarction/prevention & control , National Heart, Lung, and Blood Institute (U.S.) , Research Design , Animals , Biomarkers/blood , Cooperative Behavior , Disease Models, Animal , Drug Evaluation, Preclinical/standards , Female , Guidelines as Topic , Humans , Ischemic Preconditioning, Myocardial/standards , Male , Mice , Myocardial Infarction/blood , Myocardial Infarction/pathology , Myocardium/pathology , Predictive Value of Tests , Rabbits , Reproducibility of Results , Research Design/standards , Species Specificity , Swine , Time Factors , Troponin I/blood , United StatesABSTRACT
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.
Subject(s)
Diabetes Mellitus , Lagomorpha , MicroRNAs , Myocardial Reperfusion Injury , RNA, Long Noncoding , Animals , Humans , Rabbits , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Messenger , MicroRNAs/genetics , MicroRNAs/metabolism , Lagomorpha/genetics , Lagomorpha/metabolism , Myocardial Reperfusion Injury/drug therapy , Myocardial Reperfusion Injury/genetics , Ischemia , Proto-Oncogene Proteins c-bcl-2ABSTRACT
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 K(ATP) 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 3 weeks 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.
Subject(s)
Cardiotonic Agents/pharmacology , Janus Kinase 2/metabolism , Myocardial Reperfusion Injury/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Sirolimus/pharmacology , Animals , Apoptosis/genetics , Enzyme Activation/genetics , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Knockdown Techniques , In Vitro Techniques , Janus Kinase 2/antagonists & inhibitors , Male , Mice , Myocardial Reperfusion Injury/genetics , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type III/metabolism , Phosphorylation/drug effects , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/geneticsABSTRACT
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.
Subject(s)
Benzoates/pharmacology , Enzyme Activators/pharmacology , Guanylate Cyclase/metabolism , Hydrogen Sulfide/metabolism , Myocardial Infarction/prevention & control , Myocardial Reperfusion Injury/drug therapy , Myocytes, Cardiac/drug effects , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Apoptosis/drug effects , Cell Survival/drug effects , Cyclic GMP/metabolism , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic GMP-Dependent Protein Kinases/metabolism , Cystathionine gamma-Lyase/antagonists & inhibitors , Cystathionine gamma-Lyase/genetics , Cystathionine gamma-Lyase/metabolism , Disease Models, Animal , Enzyme Activation , Enzyme Inhibitors/pharmacology , Male , Mice , Mice, Inbred ICR , Myocardial Infarction/diagnostic imaging , Myocardial Infarction/enzymology , Myocardial Infarction/physiopathology , Myocardial Reperfusion Injury/diagnostic imaging , Myocardial Reperfusion Injury/enzymology , Myocardial Reperfusion Injury/physiopathology , Myocytes, Cardiac/enzymology , Myocytes, Cardiac/pathology , Necrosis , RNA, Messenger/metabolism , Rabbits , Soluble Guanylyl Cyclase , Ultrasonography , Up-Regulation , Ventricular Function, Left/drug effectsABSTRACT
We developed a preclinical model of myocardial ischemia/reperfusion (I/R) injury in conscious diabetic rabbits to identify an early pharmacological intervention for patients with diabetes and acute myocardial infarction (AMI). Here, we describe a reproducible protocol for induction of diabetes with subsequent manifestation of myocardial I/R injury in conscious rabbits to mimic the real-life scenario observed in clinical settings. Further, we demonstrate the efficacy of rapamycin at the onset of reperfusion to limit the adverse effect of AMI. For complete details on the use and execution of this protocol, please refer to Samidurai et al. (2020).
Subject(s)
Cardiotonic Agents/pharmacology , Diabetes Mellitus, Type 1/physiopathology , Myocardial Reperfusion Injury/etiology , Myocardial Reperfusion Injury/prevention & control , Sirolimus/pharmacology , Alloxan , Animals , Apoptosis , Balloon Occlusion/methods , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 1/complications , Disease Models, Animal , Electrocardiography , Male , Myocardial Reperfusion Injury/pathology , Rabbits , Troponin I/bloodABSTRACT
BACKGROUND: Tadalafil is a novel long-acting inhibitor of phosphodiesterase-5. Because cGMP-dependent protein kinase (PKG) signaling plays a key role in cardioprotection, we hypothesized that PKG activation with tadalafil would limit myocardial ischemia/reperfusion (I/R) injury and dysfunction. Additionally, we contemplated that cardioprotection with tadalafil is mediated by hydrogen sulfide (H(2)S) signaling in a PKG-dependent fashion. METHODS AND RESULTS: After baseline transthoracic echocardiography (TTE), adult ICR mice were injected i.p. with vehicle (10% DMSO) or tadalafil (1 mg/kg) with or without KT5823 (KT, PKG blocker, 1 mg/kg) or dl-propargylglycine (PAG, Cystathionine-gamma-lyase [CSE, H(2)S-producing enzyme] blocker; 50 mg/kg) 1 hour before coronary artery ligation for 30 minutes and reperfusion for 24 hours, whereas C57BL wild-type and CSE-knockout mice were treated with either vehicle or tadalafil. After reperfusion, TTE was performed and hearts were collected for infarct size (IS) measurement using TTC staining. Survival was increased with tadalafil (95%) compared with control (65%, P<0.05). Infarct size was reduced with tadalafil (13.2+/-1.7%) compared to vehicle (40.6+/-2.5%; P<0.05). KT and PAG abolished tadalafil-induced protection (IS: 39.2+/-1% and 51.2+/-2.4%, respectively) similar to genetic deletion of CSE (47.2+/-5.1%). Moreover, tadalafil preserved fractional shortening (FS: 31+/-1.5%) compared to control (FS: 22+/-4.8%, P<0.05). Baseline FS was 44+/-1.7%. KT and PAG abrogated the preservation of LV function with tadalafil by decline in FS to 17+/-1% and 23+/-3%, respectively. Compared to vehicle, myocardial H(2)S production was significantly increased with tadalafil and was abolished with KT. CONCLUSIONS: PKG activation with tadalafil limits myocardial infarction and preserves LV function through H(2)S signaling.
Subject(s)
Carbolines/pharmacology , Cyclic GMP-Dependent Protein Kinases/physiology , Hydrogen Sulfide/metabolism , Myocardial Reperfusion Injury/prevention & control , Phosphodiesterase 5 Inhibitors , Phosphodiesterase Inhibitors/pharmacology , Animals , Cystathionine beta-Synthase/physiology , Female , Hemodynamics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Myocardial Infarction/drug therapy , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/mortality , Tadalafil , Ventricular RemodelingABSTRACT
BACKGROUND: The most important determinant of longevity in pulmonary arterial hypertension is right ventricular (RV) function, but in contrast to experimental work elucidating the pathobiology of left ventricular failure, there is a paucity of data on the cellular and molecular mechanisms of RV failure. METHODS AND RESULTS: A mechanical animal model of chronic progressive RV pressure overload (pulmonary artery banding, not associated with structural alterations of the lung circulation) was compared with an established model of angioproliferative pulmonary hypertension associated with fatal RV failure. Isolated RV pressure overload induced RV hypertrophy without failure, whereas in the context of angioproliferative pulmonary hypertension, RV failure developed that was associated with myocardial apoptosis, fibrosis, a decreased RV capillary density, and a decreased vascular endothelial growth factor mRNA and protein expression despite increased nuclear stabilization of hypoxia-induced factor-1alpha. Induction of myocardial nuclear factor E2-related factor 2 and heme-oxygenase 1 with a dietary supplement (Protandim) prevented fibrosis and capillary loss and preserved RV function despite continuing pressure overload. CONCLUSIONS: These data brought into question the commonly held concept that RV failure associated with pulmonary hypertension is due strictly to the increased RV afterload.
Subject(s)
Blood Pressure , Heart Failure/metabolism , Hypertension, Pulmonary/metabolism , Hypertrophy, Right Ventricular/metabolism , Pulmonary Artery/metabolism , Animals , Apoptosis , Disease Models, Animal , Fibrosis , Gene Expression Regulation , Heart Failure/pathology , Heart Failure/physiopathology , Heme Oxygenase (Decyclizing)/biosynthesis , Hypertension, Pulmonary/pathology , Hypertension, Pulmonary/physiopathology , Hypertrophy, Right Ventricular/pathology , Hypertrophy, Right Ventricular/physiopathology , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Male , Pulmonary Artery/pathology , Pulmonary Artery/physiopathology , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor A/biosynthesis , Ventricular Function, RightABSTRACT
BACKGROUND: Phosphoinositide 3-kinase gamma is upregulated in the heart during acute myocardial infarction (AMI) potentially contributing to the development and maintenance of heart failure. METHODS: CD-1 male mice were randomly assigned to pharmacologic inhibition of phosphoinositide 3-kinase gamma using AS-605240 (10 mg/kg/day intraperitoneally) or vehicle (NaCl 0.9% + DMSO 25% solution) for 14 days after experimental AMI induced by surgical coronary artery ligation. Echocardiography was performed at baseline and 1, 7, 14, and 28 days after surgery to measure left ventricular dimensions and function. Infarct size was also measured at weekly intervals to evaluate for infarct resorption. RESULTS: When compared with vehicle-treated mice over the 4-week period, animals treated with AS-605240 showed a smaller increase in left ventricular cavitary dimensions, a smaller decrease in left ventricular systolic function (P < 0.05), and a significant increase in posterior wall diastolic and systolic thickness reflective of compensatory hypertrophy (P < 0.05). Initial infarct size (measured at 24 hours) was not different comparing AS-605240 (29% ± 4%) and vehicle-treated mice (31% ± 1%, P = nonsignificant). At 4 weeks after AMI, infarct size was significantly smaller in the AS-605240-treated mice (14% ± 2%) compared with vehicle-treated mice (28% ± 3%, P < 0.001), reflecting greater infarct resorption. CONCLUSIONS: Phosphoinositide 3-kinase gamma inhibition with AS-605240 after AMI leads to enhanced infarct resorption, greater compensatory hypertrophy of the nonischemic myocardium, and more favorable cardiac remodeling and function.
Subject(s)
Myocardial Infarction/drug therapy , Myocardial Infarction/enzymology , Phosphoinositide-3 Kinase Inhibitors , Quinoxalines/pharmacology , Quinoxalines/therapeutic use , Thiazolidinediones/pharmacology , Thiazolidinediones/therapeutic use , Ventricular Remodeling/drug effects , Animals , Class Ib Phosphatidylinositol 3-Kinase/metabolism , Male , Mice , Mice, Inbred ICR , Ventricular Remodeling/physiologyABSTRACT
Persistent activation of mTOR (mammalian target of rapamycin) in diabetes increases the vulnerability of the heart to ischemia/reperfusion (I/R) injury. We show here that infusion of rapamycin (mTOR inhibitor) at reperfusion following ischemia reduced myocardial infarct size and apoptosis with restoration of cardiac function in type 1 diabetic rabbits. Likewise, treatment with rapamycin protected hyperglycemic human-pluripotent-stem-cells-derived cardiomyocytes (HG-hiPSC-CMs) following simulated ischemia (SI) and reoxygenation (RO). Phosphorylation of S6 (mTORC1 marker) was increased, whereas AKT phosphorylation (mTORC2 marker) and microRNA-302a were reduced with concomitant increase of its target, PTEN, following I/R injury in diabetic heart and HG-hiPSC-CMs. Rapamycin inhibited mTORC1 and PTEN, but augmented mTORC2 with restoration of miRNA-302a under diabetic conditions. Inhibition of miRNA-302a blocked mTORC2 and abolished rapamycin-induced protection against SI/RO injury in HG-hiPSC-CMs. We conclude that rapamycin attenuates reperfusion injury in diabetic heart through inhibition of PTEN and mTORC1 with restoration of miR-302a-mTORC2 signaling.
ABSTRACT
In mouse models of cardiac disease, the type 5 (PDE5)-selective cyclic nucleotide phosphodiesterase inhibitor sildenafil has antihypertrophic and cardioprotective effects attributable to the inhibition of cGMP hydrolysis. To investigate the relevance of these findings to humans, we quantified cGMP-hydrolytic activity and its inhibition by sildenafil in cytosolic and microsomal preparations from the left ventricular myocardium of normal and failing human hearts. The vast majority of cGMP-hydrolytic activity was attributable to PDE1 and PDE3. Sildenafil had no measurable effect on cGMP hydrolysis at 10 nM, at which it is selective for PDE5, but it had a marked effect on cGMP and cAMP hydrolysis at 1 microM, at which it inhibits PDE1. In contrast, in preparations from the left ventricles of normal mice and mice with heart failure resulting from coronary artery ligation, the effects of sildenafil on cGMP hydrolysis were attributable to inhibition of both PDE5 and PDE1; PDE5 comprised approximately 22 and approximately 43% of the cytosolic cGMP-hydrolytic activity in preparations from normal and failing mouse hearts, respectively. These differences in PDE5 activities in human and mouse hearts call into question the extent to which the effects of sildenafil in mouse models are likely to be applicable in humans and raise the possibility of PDE1 as an alternative therapeutic target.
Subject(s)
Cyclic AMP/metabolism , Heart Failure/metabolism , Myocardium/metabolism , Phosphodiesterase Inhibitors/pharmacology , Piperazines/pharmacology , Sulfones/pharmacology , Animals , Coronary Vessels/physiology , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/metabolism , Cytosol/drug effects , Cytosol/metabolism , Humans , Hydrolysis , Male , Mice , Mice, Inbred ICR , Microsomes/drug effects , Microsomes/metabolism , Phosphodiesterase 3 Inhibitors , Phosphodiesterase 5 Inhibitors , Phosphodiesterase I/antagonists & inhibitors , Phosphodiesterase I/metabolism , Purines/pharmacology , Sildenafil Citrate , Subcellular Fractions/drug effects , Subcellular Fractions/metabolismABSTRACT
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.
Subject(s)
Aminobutyrates/therapeutic use , Angiotensin Receptor Antagonists/therapeutic use , Myocardial Infarction/drug therapy , Systole/physiology , Tetrazoles/therapeutic use , Ventricular Remodeling/physiology , Aminobutyrates/pharmacology , Angiotensin Receptor Antagonists/pharmacology , Animals , Biphenyl Compounds , Drug Combinations , Male , Myocardial Infarction/diagnostic imaging , Myocardial Infarction/physiopathology , Rabbits , Random Allocation , Stroke Volume/drug effects , Stroke Volume/physiology , Systole/drug effects , Tetrazoles/pharmacology , Valsartan , Ventricular Remodeling/drug effectsABSTRACT
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.
Subject(s)
Ischemic Preconditioning, Myocardial/methods , Piperazines/pharmacology , 3',5'-Cyclic-GMP Phosphodiesterases , Animals , Cyclic Nucleotide Phosphodiesterases, Type 5 , Humans , Myocardial Reperfusion Injury/prevention & control , Phosphodiesterase Inhibitors/pharmacology , Phosphodiesterase Inhibitors/therapeutic use , Phosphoric Diester Hydrolases/drug effects , Phosphoric Diester Hydrolases/pharmacology , Phosphoric Diester Hydrolases/physiology , Piperazines/therapeutic use , Purines , Sildenafil Citrate , SulfonesABSTRACT
BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: Na2S induces cardioprotective effects through miR-21-dependent attenuation of ischemic and inflammatory injury in cardiomyocytes.
Subject(s)
Hydrogen Sulfide/metabolism , MicroRNAs/genetics , Myocardial Ischemia/genetics , Animals , Cells, Cultured , Humans , Male , Mice , Mice, Inbred C57BL , MicroRNAs/metabolism , Myocardial Ischemia/immunology , Myocardial Ischemia/metabolism , Myocytes, Cardiac/immunology , Myocytes, Cardiac/metabolism , Sulfides/metabolism , Up-RegulationABSTRACT
Phosphodiesterase type-5 (PDE-5) is an enzyme that catalyzes the hydrolytic degradation of cyclic GMP - an essential intracellular second messenger that modulates diverse biological processes in living cells. Three selective inhibitors of PDE-5 - sildenafil, vardenafil and tadalafil - have been successfully used by millions of men worldwide for the treatment of erectile dysfunction. Also, sildenafil and tadalafil are currently approved for the treatment of pulmonary hypertension. Recent powerful basic science data and clinical studies suggest potential nonurological applications of PDE-5 inhibitors, including ischemia/reperfusion injury, myocardial infarction, cardiac hypertrophy, cardiomyopathy, heart failure, stroke, neurodegenerative diseases and other circulatory disorders including Raynaud's phenomenon. Future carefully controlled clinical trials would hopefully expedite their expanding therapeutic use in patients with cardiovascular disease.
ABSTRACT
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.
Subject(s)
Imidazoles/pharmacology , Myocardial Infarction/drug therapy , Myocardial Reperfusion Injury/prevention & control , Nitroglycerin/pharmacology , Piperazines/pharmacology , Potassium Channels/metabolism , Sulfones/pharmacology , Vasodilator Agents/pharmacology , Animals , Male , Mitochondria, Heart/metabolism , Mitochondria, Heart/pathology , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/pathology , Purines/pharmacology , Rabbits , Sildenafil Citrate , Triazines/pharmacology , Vardenafil DihydrochlorideABSTRACT
OBJECTIVE: To assess changes in cardiac function in animals with ischemic congestive heart failure (CHF) treated with a selective cyclo-oxygenase-2 (COX-2) inhibitor. BACKGROUND: In patients with CHF, COX-2 expression was associated with features of worsening failure. However, evidence of beneficial or detrimental functional effects of COX-2 inhibition in ischemic CHF is lacking. METHODS: Thirty male Wistar rats underwent coronary ligation and were allowed to recover for 12 months. Five sham-operated animals were used as controls. After 12 months, six surviving animals underwent baseline echocardiogram to measure end-diastolic diameter (EDD), end-systolic diameters (ESD), fractional shortening (FS), and anterior and posterior diastolic and systolic wall thicknesses. The animals were thereafter treated by daily intraperitoneal parecoxib injections (0.75 mg/kg) for 7 days. On day 7, a repeat echocardiogram was performed. RESULTS: When compared to baseline, repeat echocardiography after 7 days of parecoxib treatment showed no changes in the EDD (9.4 +/- 0.4 mm vs. 9.4 +/- 0.3 mm, P = 0.9), a significant reduction of ESD (5.5 +/- 0.8 mm vs. 6.4 +/- 0.3 mm, P = 0.028), and a significant improvement in the FS (43 +/- 3% vs. 32 +/- 5%, P = 0.027). Improvement of FS was associated with a significant change in systolic thickness in the infarct zone (3.6 +/- 0.4 mm vs. 3.0 +/- 0.1 mm, P = 0.046), whereas no significant changes in systolic thickness in the remote area were observed. CONCLUSIONS: Administration of parecoxib in ischemic CHF provides functional improvement of the peri-infarct myocardium. This finding may prove useful in improving quality of life and, perhaps, survival in patients with ischemic heart disease.
Subject(s)
Cyclooxygenase 2 Inhibitors/therapeutic use , Heart Failure/prevention & control , Isoxazoles/therapeutic use , Myocardial Ischemia/complications , Myocardium , Animals , Cyclooxygenase 2/biosynthesis , Cyclooxygenase 2 Inhibitors/administration & dosage , Cyclooxygenase 2 Inhibitors/pharmacology , Disease Models, Animal , Echocardiography , Heart Failure/etiology , Heart Function Tests , Isoxazoles/administration & dosage , Isoxazoles/pharmacology , Male , Myocardium/enzymology , Myocardium/pathology , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Cyclooxygenase 2 Inhibitors/therapeutic use , Isoxazoles/therapeutic use , Myocardial Infarction/drug therapy , Ventricular Remodeling/drug effects , Animals , Apoptosis/drug effects , Arterioles/drug effects , Arterioles/pathology , Arterioles/physiopathology , Cyclooxygenase 2 Inhibitors/pharmacology , Cytokines/blood , Echocardiography , Heart/drug effects , Heart/physiopathology , Isoxazoles/pharmacology , Male , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardium/pathology , Organ Size/drug effects , Rats , Rats, Wistar , Survival AnalysisABSTRACT
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.