Nicorandil Pretreatment Research Articles

Direct and indirect activation of the adenosine triphosphate-sensitive potassium channel to induce spinal cord ischemic metabolic tolerance.

The mitochondrial adenosine triphosphate-sensitive potassium channel is central to pharmacologically induced tolerance to spinal cord injury. We hypothesized that both direct and nitric oxide-dependent indirect activation of the adenosine triphosphate-sensitive potassium channel contribute to the induction of ischemic metabolic tolerance. Spinal cord injury was induced in adult male C57BL/6 mice through 7minutes of thoracic aortic crossclamping. Pretreatment consisted of intraperitoneal injection 3 consecutive days before injury. Experimental groups were sham (no pretreatment or ischemia, n=10), spinal cord injury control (pretreatment with normal saline, n=27), Nicorandil 1.0mg/kg (direct and indirect adenosine triphosphate-sensitive potassium channel opener, n=20), Nicorandil 1mg/kg+carboxy-PTIO 1mg/kg (nitric oxide scavenger, n=21), carboxy-PTIO (n=12), diazoxide 5mg/kg (selective direct adenosine triphosphate-sensitive potassium channel opener, n=25), and DZ 5mg/kg+carboxy-PTIO 1mg/kg, carboxy-PTIO (n=23). Limb motor function was assessed using the Basso Mouse Score (0-9) at 12-hour intervals for 48hours after ischemia. Motor function was significantly preserved at all time points after ischemia in the Nicorandil pretreatment group compared with ischemic control. The addition of carboxy-PTIO partially attenuated Nicorandil's motor-preserving effect. Motor function in the Nicorandil+carboxy-PTIO group was significantly preserved compared with the spinal cord injury control group (P<.001), but worse than in the Nicorandil group (P=.078). Motor preservation in the diazoxide group was similar to the Nicorandil+carboxy-PTIO group. There was no significant difference between the diazoxide and diazoxide+carboxy-PTIO groups. Both direct and nitric oxide-dependent indirect activation of the mitochondrial adenosine triphosphate-sensitive potassium channel play an important role in pharmacologically induced motor function preservation.

Nicorandil inhibits TLR4/MyD88/NF-κB/NLRP3 signaling pathway to reduce pyroptosis in rats with myocardial infarction.

Pyroptosis is an inflammatory cell death that regulates cardiomyocyte loss after myocardial infarction. Reports indicate that nicorandil has a strong anti-inflammatory effect and protects the myocardium from myocardial infarction. However, its relationship with pyroptosis is largely unreported. Here, we investigated to influence and mechanism of action of nicorandil on cardiomyocyte pyroptosis. Forty Sprague Dawley rats were randomly assigned to sham, MI, MI + nicorandil, and MI + nicorandil + TAK242 groups (10 per group). Myocardial infarction modeling was performed through ligation of the anterior descending branch of the left coronary artery. The function of cardiac was evaluated through echocardiography, detection of myocardial adenine nucleotides, cTnI, LDH, TTC, and HE staining. Moreover, we used qRT-PCR, immunohistochemistry, and Western blotting to examine the expression of pyroptosis-related molecules and the inflammasome pathway of TLR4/MyD88/NF-κB/NLRP3. Myocardial infarction caused the activation of GSDMD, aggravated myocardial injury, and triggered cardiac dysfunction. Myocardial infarction induced pyroptotic cell death, manifested as upregulation in mRNA and protein levels associated with pyroptosis, including caspase-1 cleavage and increased expression of IL-1β and IL-18. These changes were mitigated by nicorandil. The achieved data implicate that myocardial infarction induces pyroptosis via the TLR4/MyD88/NF-κB/NLRP3 pathway, which can be inhibited by nicorandil pretreatment. Therefore, nicorandil exerts cardioprotective effects by activating KATP channels, and at least in part through inhibition of the TLR4/MyD88/NF-κB/NLRP3 pathway to reduce myocardial infarction-induced pyroptosis. As such, it is a potential therapy for ischemic heart disease.

Optimizing Nicorandil for Spinal Cord Protection in a Murine Model of Complex Aortic Intervention

There are currently no clinically utilized pharmacological agents for the induction of metabolic tolerance to spinal cord ischemia-reperfusion injury in the setting of complex aortic intervention. Nicorandil, a nitric oxide donor and ATP-sensitive potassium (KATP) channel opener, has shown promise in neuroprotection. However, the optimized clinical application of the drug and its mechanism of neuroprotection remains unclear. We hypothesized that 3-days pretreatment would confer the most effective neuroprotection, mediated by mitochondrial KATP channel activation. Spinal cord injury was induced by 7 minutes of thoracic aortic cross-clamping in adult male C57BL/6 mice. Time course: mice received 0.1 mg/kg nicorandil for 10 min, 4 hours, and 3 consecutive days prior to ischemia compared with control. Dose challenge: mice received 3-days nicorandil pretreatment comparing 0.1 mg/kg, 1.0 mg/kg, 5.0 mg/kg, and saline administration. Mitochondrial KATP channel blocker 5-hydroxy-decanoate (5HD) was co-administered to elucidate mechanism. Limb motor function was evaluated, and viable anterior horn neurons quantified. Nicorandil pretreatment at 4 hours and 3 days before ischemia demonstrated significant motor function preservation; administration 10 minutes before ischemia showed no neuroprotection. All nicorandil doses showed significant motor function preservation. Three days administration of Nicorandil 1.0 mg/kg was most potent. Neuroprotection was completely abolished by 5HD co-administration. Histological analysis showed significant neuron preservation with nicorandil pretreatment, which was attenuated by 5HD co-administration. Three days administration of Nicorandil 1.0 mg/kg showed near-total motor function preservation in a murine spinal cord ischemia-reperfusion model, mediated by the mitochondrial KATP channel.

Abstract 13107: Nitroglycerin Pretreatment Induces Metabolic Tolerance to Spinal Cord Ischemia-reperfusion Injury Through Nitric Oxide-mediated Mitochondrial Katp Channel Activation

Introduction: Pharmacologic induction of metabolic tolerance to spinal cord ischemia-reperfusion injury (SCI) after thoracoabdominal aortic intervention is not well established. We previously demonstrated that nicorandil pretreatment preserved motor function in a murine SCI model via direct mitochondrial ATP-sensitive potassium (KATP) channel activation and nitric oxide (NO) donation. However, the independent role of NO-mediated neuroprotection has not been elucidated. Hypothesis: Nitroglycerin pretreatment will induce neuroprotection through NO-mediated KATP channel activation. Methods: SCI was induced by 7 minutes of thoracic aortic cross-clamping in adult male C57BL/6 mice. Pretreatment constituted intraperitoneal injection 3 consecutive days before injury. Experimental groups: sham (no pretreatment or ischemia, n=10), SCI control (normal saline, n=20), nitroglycerin 1 mg/kg (n=18), nitroglycerin 1 mg/kg + 5-hydroxydecanonate 5 mg/kg (5HD, mitochondrial KATP channel blocker, n=13), 5HD 5 mg/kg (n=10), nitroglycerin 1 mg/kg + carboxy-PTIO (cPTIO) 1 mg/kg (NO scavenger, n=16), and cPTIO 1 mg/kg (n=10). Limb motor function and the number of viable neurons within the anterior horn of the spinal cord were evaluated. Results: Compared to SCI control, motor function was significantly preserved in the nitroglycerin pretreatment group at every time point after ischemia. In the nitroglycerin+5HD and nitroglycerin+cPTIO groups, motor preservation was significantly attenuated compared to nitroglycerin pretreatment (p&lt;0.001). Histological analysis showed significant neuron preservation in the nitroglycerin pretreatment group compared with SCI control (p=0.011). This preservation was completely attenuated with 5HD or cPTIO co-administration (p=0.001). Conclusions: Nitroglycerin pretreatment significantly preserved motor function in a murine SCI model through NO-mediated KATP channel activation.

Reactive Oxygen Species Mediate Nicorandil-induced Metabolic Tolerance to Spinal Cord Injury

Spinal cord injury remains a devastating complication of thoracoabdominal aortic surgery. We previously demonstrated that pretreatment with nicorandil preserved motor function in a murine spinal cord injury model through mitochondrial adenosine triphosphate-sensitive potassium channel activation. We hypothesized that the neuroprotective effect of nicorandil is mediated by downstream generation of reactive oxygen species. Spinal cord injury was induced by 7 minutes of thoracic aortic cross-clamping in adult male C57BL/6 mice. Five groups were evaluated: ischemic control (n= 19); nicorandil 1.0 mg/kg (n= 17); nicorandil 1.0 mg/kg plus N acetyl L-cysteine (NAC [reactive oxygen species scavenger, n= 18)]) 150 mg/kg; NAC 150 mg/kg (n= 13); and sham (n= 10). Limb motor function and the number of viable neurons within the anterior horn of the spinal cord were evaluated. Mice in the sham group showed no functional deficits after surgery. Compared with ischemic control, motor function was significantly preserved in the nicorandil pretreatment group at every timepoint after ischemia. In the nicorandil plus NAC group, the motor-preserving effect of nicorandil was completely abolished (P < .001). Viable neuron quantification showed significant neuron preservation in the nicorandil group (29.± 2.6) compared with the ischemic control group (18.5 ± 2.1, P= .024) and nicorandil plus NAC group (14 ± 8.3, P= .001); no significant difference was observed between the ischemic control group and nicorandil plus NAC group (P= 0.768). Reactive oxygen species generation plays a key role in the nicorandil-induced metabolic tolerance to spinal cord injury. Manipulation of mitochondrial adenosine triphosphate-sensitive potassium channels may lead to improvement in preventing spinal cord injury after thoracoabdominal aortic interventions.

Role of nitric oxide donor in methotrexate-induced testicular injury via modulation of pro-inflammatory mediators, eNOS and P-glycoprotein.

Methotrexate (MTX) is a widely used chemotherapeutic agent but its clinical use is challenged with different forms of toxicities including testicular injury. The aim of the current study was to evaluate the potential protective effect of potassium channel opener, nicorandil (NIC) (3 and 10 mg/kg/day) on MTX-induced testicular injury in a rat model. Rats were randomly divided into four groups (nine rats each) and treated for 2 weeks as follows: (I) normal control (CON group) received vehicle, (II) model group (MTX group) given MTX (20 mg/kg) single intraperitoneal (i.p.) injection dose on 11th day, (III) MTX + NLD group treated with NIC (3 mg/kg/day) orally for 2 weeks and MTX (20 mg/kg) single i.p. dose on 11th day, and (IV) MTX + NHD group treated with NIC (10 mg/kg/day) orally for 2 weeks and MTX (20 mg/kg) single i.p. injection on the 11th day. The testicular injury was assessed biochemically via serum testosterone, total antioxidant capacity, testicular oxidative stress parameters, P-glycoprotein, tumor necrosis factor-alpha, and interleukin-1β. Furthermore, histopathological evaluation, endothelial nitric oxide synthase (eNOS) immunoexpression, and detection of p53 expression level using Western blotting were performed. Results showed that MTX induced testicular injury which was proved by both biochemical and histopathological evaluations. Our results concluded that NIC pretreatment attenuated MTX-induced testicular injury via significantly increased eNOS immunoexpression, antiapoptotic, anti-inflammatory, and antioxidant properties. Interestingly, NIC high dose is more protective than low dose.

The impact of oral nicorandil pre-treatment on ST resolution and clinical outcome of patients with acute ST-segment elevation myocardial infarction undergoing primary coronary angioplasty: A randomized placebo controlled trial.

Introduction: Literature has shown the effects of intravenous/intracoronary nicorandil on increased myocardial salvage in patients with ST-segment elevation myocardial infarction (STEMI) treated with mechanical reperfusion. However, the possible cardioprotective effect of oral nicorandil on the clinical outcome prior to primary coronary angioplasty is not well documented. Our aim was to assess the effect of oral nicorandil on primary percutaneous coronary intervention (PPCI). Methods: A total of 240 patients with acute STEMI undergoing PPCI were randomly assigned to oral nicorandil (Intervention, n=116) and placebo (Control, n=124) groups. The intervention group received 20 mg oral nicorandil at the emergency department and another 20 mg oral nicorandil in the catheterization laboratory just before the procedure. The control group received matched placebo. Our primary outcome was ST-segment resolution ≥50% one hour after primary angioplasty. Secondary outcome was in-hospital major adverse cardiovascular events (MACE), defined as a composite of death, ventricular arrhythmia, heart failure and stroke. Results: In the patients of intervention and control groups, the occurrence of ST-segment resolution ≥ 50% were 68.1% and 62.9% respectively, (P =0.27). In-hospital MACE occurred less frequently in the intervention group, compared to placebo group (11.2% vs. 22.5%, P =0.012). Conclusion: Although the administration of oral nicorandil before primary coronary angioplasty did not improve ST-segment resolution in patients with acute STEMI, its promoting effects was remarkable on in-hospital clinical outcomes. Clinical Registration: IRCT20140512017666N1

Nicorandil prevents doxorubicin-induced human umbilical vein endothelial cell apoptosis

Nicorandil is an adenosine triphosphate-sensitive potassium channel opener with additional antioxidant properties. Doxorubicin (DOX) is an anticancer drug that exerts oxidation-mediated adverse cardiovascular effects. This study examined the effects of nicorandil on DOX-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs) and underlying intracellular signaling mechanisms. Cultured HUVECs were pretreated with nicorandil (0.1, 0.3, 1, 3, and 10 μM) for 12 h and then treated with DOX (1 μM) for 24 h. Cell viability and cytotoxicity were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, respectively. Cell apoptosis was examined using a caspase-3 activity assay, and DNA fragmentation was detected through TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining. Western blot analysis was conducted to determine the related protein expression. DOX markedly increased reactive oxygen species production, p53 expression, caspase-3 activity, cleaved caspase-3 levels, and TUNEL-positive cell numbers but reduced Bcl-2 expression and intracellular antioxidant enzyme levels; these effects were effectively antagonized through nicorandil (3 μM, 12 h) pretreatment, which resulted in HUVECs being protected from DOX-induced apoptosis. Activating transcription factor 3 (ATF3), a stress-induced transcription factor, was induced by nicorandil (3 μM). Furthermore, nicorandil (3 μM) enhanced nuclear factor erythroid 2–related factor 2 (Nrf2) translocation and heme oxygenase-1 (HO-1) expression. ATF3 short interfering RNA significantly attenuated nicorandil-mediated Nrf2 translocation, HO-1 expression, and inhibitory effects on DOX-stimulated reactive oxygen species production and cell apoptosis. In summary, nicorandil may protect HUVECs from DOX-induced apoptosis, in part through ATF3-mediated Nrf2/HO-1 signaling pathways, which potentially protect the vessels from severe DOX toxicity.

Nicorandil alleviated cardiac hypoxia/reoxygenation-induced cytotoxicity via upregulating ketone body metabolism and ACAT1 activity.

To study the effect of nicorandil pretreatment on ketone body metabolism and Acetyl-CoA acetyltransferase (ACAT1) activity in hypoxia/reoxygenation (H/R)-induced cardiomyocytes. In our study, we applied H9c2 cardiomyocytes cell line to evaluate the cardioprotective effects of nicorandil. We detected mitochondrial viability, cellular apoptosis, reactive oxygen species (ROS) production and calcium overloading in H9c2 cells that exposed to H/R-induced cytotoxicity. Then we evaluated whether nicorandil possibly regulated ketone body, mainly β-hydroxybutyrate (BHB) and acetoacetate (ACAC), metabolism by regulating ACAT1 and Succinyl-CoA:3-keto-acid coenzyme A transferase 1 (OXCT1) protein and gene expressions. Nicorandil protected H9c2 cardiomyocytes against H/R-induced cytotoxicity dose-dependently by mitochondria-mediated anti-apoptosis pathway. Nicorandil significantly decreased cellular apoptotic rate and enhanced the ratio of Bcl-2/Bax expressions. Further, nicorandil decreased the production of ROS and alleviated calcium overloading in H/R-induced H9c2 cells. In crucial, nicorandil upregulated ACAT1 and OXCT1 protein expressions and either of their gene expressions, contributing to increased production of cellular BHB and ACAC. Nicorandil alleviated cardiomyocytes H/R-induced cytotoxicity through upregulating ACAT1/OXCT1 activity and ketone body metabolism, which might be a potential mechanism for emerging study of nicorandil and other KATP channel openers.

Effects of pretreatment with nicorandil on PDCD4/NF-κB/TNF-α pathway in coronary microembolization

Objective To investigate the role of nicorandil pretreatment on protecting myocardium after coronary microembolization (CME) and on the PDCD4/NF-κB/TNF-α signaling pathway in miniature pigs. Methods Fifteen Bama miniature pigs were randomly(random number) divided into the sham operation group (sham group), microembolization group (CME group) and CME plus nicorandil group, with 5 pigs in each group. The CME model was constructed by injecting polyethylene microspheres via microcatheter into the left anterior descending artery, and pigs in the sham group were injected with the same amount of saline. Pigs in the CME plus nicorandil group were injected intravenously with nicorandil (150 μg/kg) via ear vein 30 min before CME. Cardiac function indexes were measured using cardiac ultrasonography. The expression of PDCD4 and TNF-α mRNA in myocardium were detected by fluorescence quantitative PCR, and the protein expression of PDCD4 and TNF-α in myocardium were detected by Western blotting. NF-κB activation was evaluated by electrophoretic mobility shift assay. Results (1) Cardiac function was significantly lower and the level of serum cTnI was significantly higher in the CME group compared with the sham group. CME reduced myocardial systolic dysfunction and left ventricular dilatation. The CME plus nicorandil group showed improved CME-induced cardiac function and reduced serum cTnI level when compared with the CME Group (P < 0.05). (2) Compared with the CME group, the CME plus nicorandil group showed lower PDCD4 and TNF-α expression and NF-κB activity as well as improved cardiac function (P < 0.05). Conclusions The pretreatment of nicorandil effectively reduced the myocardial damage caused by CME, mainly through inhibiting the PDCD4/NF-κB/TNF-α pathway in cardiomyocytes. Key words: Coronary vessels; Embolism; Nicorandil; Programmed cell death 4; Nuclear facotr-κappa B; Tumor necrosis factor α; Inflammation; Myocardial damage

Nicorandil pretreatment inhibits myocardial apoptosis and improves cardiac function after coronary microembolization in rats.

BackgroundNicorandil (NIC) is a vasodilatory drug used to treat angina. However, its efficacy of cardioprotection in coronary microembolization (CME) is largely unknown. This study was undertaken to determine whether nicorandil pretreatment could attenuate myocardial apoptosis and improve cardiac function after CME in rats.MethodsForty-five rats were randomly divided into a Sham group, a CME group and a CME + NIC (NIC) group (n = 15 per group). CME was established by injecting plastic microspheres (42 µm in diameter) into the left ventricle of the rats in all of the groups except the Sham group. The NIC group received nicorandil at 3 mg/kg per day for seven days before the operation. Cardiac function was assessed by echocardiography, the expression levels of cleaved caspase-9/8/3 were detected by Western blot, microinfarction area was measured by haematoxylin-basic fuchsin picric acid staining, and myocardial apoptosis was detected by TUNEL staining.ResultsCompared to that in the Sham group, cardiac function in the CME group was significantly decreased (P < 0.05). However, compared to the CME group, the NIC group showed improved cardiac function (P < 0.05). The expression levels of cleaved caspase-9/8/3 protein and myocardial apoptosis were dramatically increased in the CME group compared to those in the Sham group (P < 0.05), while the NIC pretreatment group had significantly decreased expression levels of cleaved caspase-9/8/3 protein as well as a decreased apoptotic rate (P < 0.05).ConclusionsNIC pretreatment inhibited CME-induced myocardial apoptosis and improved cardiac function through blockade of the mitochondrial and death receptor-mediated apoptotic pathways.

Role of TLR4/MyD88/NF-κB signaling pathway in coronary microembolization-induced myocardial injury prevented and treated with nicorandil

Coronary microembolization (CME) is a common complication during the treatment of acute coronary syndrome (ACS) and percutaneous coronary intervention (PCI). Nicorandil can be used to prevent myocardial injury after PCI to reduce the incidence of coronary no-reflow and slow flow, and play a role in myocardial protection, suggesting that its mechanism may be related to the inhibition of CME-induced inflammation of cardiomyocytes. However, the specific mechanism remains unclear. This study investigated the myocardial protective effects of nicorandil pretreatment on CME-induced myocardial injury and the specific mechanism of its inhibition of myocardial inflammation. An CME rat model exhibited CME-induced myocardial inflammation and the elevation of serum tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β based on echocardiography, myocardial enzyme detection, hematoxylin and eosin (HE) and hematoxylin-basic fuchsin-picric acid (HBFP) stainings, ELISA, quantitative real-time PCR, and western blotting. Nicorandil treatment seven days before CME induction effectively inhibited myocardial inflammation, ameliorated myocardial injury, and improved cardiac function, mainly by inhibiting Toll-like receptor 4 (TLR4)-mediated myeloid differentiation primary response protein 88 (MyD88)-dependent nuclear factor-kappa B (NF-κB) signaling. Rat neonatal cardiomyocyte experiments further confirmed that nicorandil ameliorated lipopolysaccharide (LPS)-induced myocardial inflammation and improved cardiomyocyte survival. The specific mechanisms mainly involved the inhibition of TLR4/MyD88/NF-κB signaling and the reduction of the inflammatory cytokines TNF-α and IL-1β released from cardiomyocytes. In summary, nicorandil significantly protected cardiomyocytes from CME-induced myocardial injury mainly by inhibiting TLR4/MyD88/NF-κB signaling, thereby reducing the onset of CME-induced myocardial inflammation. This could be one of the important mechanisms for reducing postoperative myocardial injury via PCI-preoperative prophylactic treatment with nicorandil.

The protective effect of nicorandil on cardiomyocyte apoptosis after coronary microembolization by activating Nrf2/HO-1 signaling pathway in rats

BackgroundMyocardial apoptosis is considered to be the chief cause of progressive cardiac dysfunction induced by coronary microembolization (CME), and the Nrf2/HO-1 signaling pathway is involved in CME-induced myocardial apoptosis. Nicorandil (NIC) has multiple beneficial cardiovascular effects on myocardial injury. Therefore, this study was undertaken to analyze the role of NIC pretreatment in the inhibiting myocardial apoptosis after CME in rats. MethodsForty rats were divided into Sham group, CME group, CME plus NIC (NIC) group, and CME plus AAV9-Nrf2 (AAV9-Nrf2) group (n = 10 per group). CME-induced myocardial apoptosis model was established through injecting plastic microspheres (42 μM) into the left ventricle except the Sham group. NIC group received nicorandil 3 mg/(kg.d) for 7 days before the operation. Cardiac function was assessed by echocardiography. The mRNA expression level of Nrf2 was detected by RT-PCR. The protein expression levels of Nrf2, HO-1, Bcl-2, Bax and cleaved caspase-3 were detected by Western blot. The size of the microinfarction area was measured by HBFP staining; myocardial apoptosis was analyzed by TUNEL staining. ResultsCompared with the sham group, the cardiac function and the expression level of Nrf2, HO-1 and Bcl-2were decreased, while myocardial apoptosis and the expression of Bax and cleaved caspase-3 were increased in the CME group. Compared with the CME group, cardiac function was significantly improved, the expression levels of Nrf2, HO-1, and Bcl-2 were increased, the expression of Bax and cleaved caspase-3 were decreased, and the myocardial apoptosis was attenuated in the NIC group and AAV9-Nrf2 group. ConclusionNIC pretreatment effectively inhibit CME-induced myocardial apoptosis and improve cardiac function. The protective effects are mediated through the activation of the Nrf2/HO-1 signaling in cardiomyocytes.

Effects of nicorandil on PI3K/Akt signaling pathway and its anti-apoptotic mechanisms in coronary microembolization in rats.

Coronary microembolization (CME) is a common complication of percutaneous coronary intervention (PCI) for acute coronary syndrome. It leads to myocardial apoptosis and cardiac dysfunction. Nicorandil pretreatment can prevent PCI-related myocardial injury and reduce the incidence of no- or slow-reflow phenomena. This cardioprotective effect is probably attributable to the suppression of CME-induced cardiomyocyte apoptosis, but the specific mechanisms have not been clarified. We aimed to investigate the protective effects of nicorandil pretreatment on CME-induced myocardial injury and clarify the underlying mechanisms. In vivo studies, we used echocardiography, cardiac-enzymes measurement, hematoxylin–basic fuchsin–picric acid staining, TUNEL assay, and western blot, and found that CME significantly increased apoptotic cardiomyocytes in the infarct and peri-infarct areas in rats. The PI3K/Akt signaling pathway was involved in cardiomyocyte apoptosis. Nicorandil pretreatment given 7 days before CME effectively reduced cardiomyocyte apoptosis and myocardial injuries in rats, mainly through the activation of PI3K/Akt signaling. In vitro studies further showed that nicorandil reduced hypoxia-induced cardiomyocyte apoptosis and improved cardiomyocyte-survival rate. The PI3K-specific inhibitor LY294002 reduced these cardioprotective effects, indicating that they were attributable to the activation of the PI3K/Akt signaling pathway. In conclusion, nicorandil has significant cardioprotective effects in CME mainly through the activation of the PI3K/Akt signaling pathway and reduction of CME-induced cardiomyocyte apoptosis. Our findings may provide important support for the pre-PCI use of nicorandil to reduce post-PCI myocardial injuries.

Vascular calcification abrogates the nicorandil mediated cardio-protection in ischemia reperfusion injury of rat heart

The present study was aimed to determine the efficacy of nicorandil in treating cardiac reperfusion injury with an underlying co-morbidity of vascular calcification (VC). Adenine diet was used to induce VC in Wistar rat and the heart was isolated to induce global ischemia reperfusion (IR) by Langendorff method, with and without the nicorandil (7.5mg/kg) pre-treatment and compared with those fed on normal diet. The adenine-treated rats displayed abnormal ECG changes and altered mitochondrial integrity compared to a normal rat heart. These hearts, when subjected to IR increased the infarct size, cardiac injury (measured by lactate dehydrogenase and creatine kinase activity in the coronary perfusate) and significantly altered the hemodynamics compared to the normal perfused heart. Nicorandil pretreatment in rat fed on normal diet enhanced the hemodynamics significantly (P<0.05) along with a substantial reduction in the mitochondrial dysfunction (measured by high ADP to oxygen consumption ratio, respiratory control ratio, enzyme activities and less swelling behavior) when subjected to IR. However, this cardio-protective effect of nicorandil was absent in rat heart with underlying calcification. Our results suggest that, the protective effect of nicorandil, a known mitochondrial ATP linked K+ channel opener, against myocardial reperfusion injury was confined to normal rat heart.

Nicorandil, a Nitric Oxide Donor and ATP-Sensitive Potassium Channel Opener, Protects Against Dystrophin-Deficient Cardiomyopathy.

Dystrophin-deficient cardiomyopathy is a growing clinical problem without targeted treatments. We investigated whether nicorandil promotes cardioprotection in human dystrophin-deficient induced pluripotent stem cell (iPSC)-derived cardiomyocytes and the muscular dystrophy mdx mouse heart. Dystrophin-deficient iPSC-derived cardiomyocytes had decreased levels of endothelial nitric oxide synthase and neuronal nitric oxide synthase. The dystrophin-deficient cardiomyocytes had increased cell injury and death after 2 hours of stress and recovery. This was associated with increased levels of reactive oxygen species and dissipation of the mitochondrial membrane potential. Nicorandil pretreatment was able to abolish these stress-induced changes through a mechanism that involved the nitric oxide-cyclic guanosine monophosphate pathway and mitochondrial adenosine triphosphate-sensitive potassium channels. The increased reactive oxygen species levels in the dystrophin-deficient cardiomyocytes were associated with diminished expression of select antioxidant genes and increased activity of xanthine oxidase. Furthermore, nicorandil was found to improve the restoration of cardiac function after ischemia and reperfusion in the isolated mdx mouse heart. Nicorandil protects against stress-induced cell death in dystrophin-deficient cardiomyocytes and preserves cardiac function in the mdx mouse heart subjected to ischemia and reperfusion injury. This suggests a potential therapeutic role for nicorandil in dystrophin-deficient cardiomyopathy.

Abstract 213: Dystrophic iPSC-derived Cardiomyocytes Have Mislocalization of eNOS and Increased Susceptibility to Cell Death which is Reversed by the Nitrate-like Properties of Nicorandil.

Background: Cardiomyopathy is a leading cause of death in Duchenne and Becker muscular dystrophy (D/BMD) patients and specific therapies are lacking. Dystrophin deficiency is associated with mislocalization of nitric oxide synthase (NOS) whose dysregulation contributes to the pathogenesis of dystrophic cardiomyopathy. We have modeled dystrophic cardiomyopathy using patient-specific induced pluripotent stem cells (iPSCs) and show that DMD- and BMD- iPSC derived cardiomyocytes have mislocalization of endothelial (eNOS) but not neuronal NOS (nNOS). In this study, we determined whether nicorandil, a drug with nitrate-like properties, protects against stress-induced dystrophic cardiomyocyte injury. Methods/Results: DMD-iPSC (exon 3-6 deletion), BMD-iPSC (exon 45-53 deletion) and non-dystrophic (N)-iPSC lines were differentiated into cardiomyocytes (iCMs) and matured for 35-38 days. Immunofluorescence revealed eNOS co-localized with dystrophin along the sarcolemma in N-iCMs but not in DMD- or BMD-iCMs. eNOS also co-localized with caveolin-1 in all groups. nNOS did not co-localize with dystrophin but with the ryanodine receptor 2 in all groups. Both DMD- and BMD-iCMs have increased cell death as determined by TUNEL + staining when subject to 2 hours of metabolic stress and 4 hours of recovery (DMD: 66±3%, BMD: 29±4% vs. 1.5±0.04%; n=3-6). This was associated with dissipation of the mitochondrial membrane potential as monitored by laser-scanning confocal microscopy (DMD: complete loss, BMD: 441±168 vs. N: 891±183 AU; n=3-5). Nicorandil pretreatment was able to restore mitochondrial membrane potential and decrease cell death. The protective effects of nicorandil were abolished by ODQ, a selective inhibitor of NO-sensitive guanylyl cyclase. Conclusion: Nicorandil protects against stress-induced cell death and preserves mitochondrial function in dystrophic cardiomyocytes through its nitrite-like properties. This suggests a potential therapeutic role of nicorandil in the treatment of dystrophic cardiomyopathy.

Effect of nicorandil pretreatment on myocardial mitochondria in a rabbit model of myocardial ischemia-reperfusion

Objective To investigate the effect of nicorandil pretreatment on myocardial mitochondria in a rabbit model of myocardial ischemia-reperfusion (I/R). Methods Tirty-two healthy male New Zealand white rabbits weighing 2.0-2.5 kg aged 4 months were randomly allocated into 4 groups ( n = 8 each): Ⅰ group sham operation (group S); Ⅱ group I/R; Ⅲ group nicorandil pretreatment (group N) and Ⅳ group nicorandil + 5 hydroxydecanoic acid (group N + 5-HD). Myocardial I/R was induced by 30 min occlusion of left circumflex coronary artery followed by 120 min reperfusion. In group N and N + 5-HD a bolus of nicorandil 100 μg/kg was given iv at 10 min before myocardial ischemia followed by continuous infusion at 10 μg· kg-1 · min-1 until the beginning of myocardial ischemia. In group Ⅳ a bolus of 5-HD 5 mg/kg was injected iv at 20 min before myocardial ischemia.The animals were sacrificed at the end of 120 min reperfusion. The mitochondrial membrane potential was measured by flow cytometry using JC-1 fluorescence probe as indicator. Bcl-2, Bax and cytochrome c protein expression was determined by immuno-histochemistry. Myocardial ultrastructure was examined with transmission electron microscope. Results Red fluovescence intensity indicating normal live cells was significantly higher, the green fluorescence intensity indicating apoptotic cells was lower and red/green fluorescence intensity ratio was higher; the Bcl-2/Bax ratio was significantly higher and cytochrome c protein expression lower in group N than in group I/R.5-HD administration negated the protective effect of nicorandil pretreatment against myocardial I/R injury. Conclusion Nicorandil stabilizes mitochondrial membrane potential, decreases cytochrome c protein releasing, and suppresses mitochondrial apoptotic signal transduction by opening the mito-KATP channels. Key words: Nicorandil; Myocardial reperfusion injury; Mitochondria,heart

Gastroprotective Effect of Nicorandil in Indomethacin and Alcohol-Induced Acute Ulcers

Despite the fact that dietary habits and lifestyles are incredibly advancing, gastric ulceration is still a terrible complaint. Extensive use of non-steroidal anti-inflammatory drugs (NSAIDs) and alcohol, in addition to stress, are all predisposing factors for ulcers. Most medical treatments are always time consuming and not efficient or satisfactory to the patients. Cardiovascular patients always need NSAIDs, or mostly cannot quit alcohols, while using many cardiovascular drugs. We aim to study a possible benefit of a common nitrogen oxide donor, anti-anginal drug, nicorandil [N-(2-hydroxyethyl) nicotinamide nitrate ester], in managing acute gastric ulcers through studying its effect on some relevant intermediates to ulcerogenesis as lipid peroxidation, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO). In addition, gastric mucosal histology was studied to pursue the drug effects on tissue level. Our study revealed that both indomethacin and alcohol induced gastric ulcer mainly through up-regulation of gastric mucosal lipid peroxidation, local tissue inflammation, leukocytic infiltration, and necrosis. Both ulcerogens significantly elevated TNF-alpha and decreased NO, initiating ulcer formation. Nicorandil pretreatment depicted a higher preventive index in indomethacin- (89.8%) and alcohol-induced (77.7%) acute ulceration. On the tissue level, it also protected the gastric mucosa combating leukocyte infiltration and tissue congestion. Nicorandil protected tissue necrosis through decreasing oxidative stress, elevating NO levels, and down-regulating the ulcerogen-induced TNF-alpha elevation and improved sub-mucosal blood supply. We conclude that nicorandil may be a suitable bimodal treatment for cardiovascular patients who are at high risk of gastric ulcers by using variable analgesics to alleviate possible cardiac pain episodes, and probably frequent doses will offer a more established and long-lasting protection.

Nicorandil suppresses the increases in plasma level of matrix metalloproteinase activity and attenuates left ventricular remodeling in patients with acute myocardial infarction

Nicorandil, a hybrid KATP channel opener and nicotinamide nitrate, reduces no-reflow phenomenon and improves cardiac function in patients with acute myocardial infarction (AMI). We reported that nicorandil suppresses radical formation in patients with AMI undergoing primary percutaneous coronary intervention (PCI). In the present study, we tested the hypothesis that nicorandil treatment suppresses MMP activities and predicts ventricular remodeling in AMI. Sixty-two patients with AMI were randomized into nicorandil pretreatment (n = 31) and control (n = 31) groups after admission and underwent primary PCI. Nicorandil was administered as a bolus injection (4 mg) followed by constant infusion (8 mg/h) for 24 h just after admission. On days 1, 2, and 14 after the onset of AMI, the plasma levels of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by enzyme-linked immunosorbent assay and the activities by gelatin zymography. There were no differences in the baseline clinical characteristics between the two groups. On day 1, there were no differences in both MMP-2 and MMP-9 levels and their activities between the two groups. However, both MMP-2 and MMP-9 levels and their activities were significantly lower in nicorandil than in control group on day 2 (MMP-2 level, 1 014 +/- 39 vs 1 174 +/- 44 ng/ml; MMP-9 level, 17 +/- 1 vs 23 +/- 2 ng/ml; both P < 005) and on day l4 (MMP-2 level, 970 +/- 38 vs 1 221 +/- 44 ng/ml; MMP-9 level, 17 +/- 1 vs 23 +/- 1 ng/ml; both P < 0.05). Left ventricular end-diastolic volume index (LVEDVI) at acute phase was not different between the two groups. At 6 months after AMI, LVEDVI was significantly smaller in nicorandil than in the control group (83 +/- 4 vs 96 +/- 4 ml/m2, P < 0.05). The change in LVEDVI from acute phase to 6 months was positively correlated with MMP-2 and MMP-9 levels and activities. Nicorandil suppresses the increases in MMP levels and activities and prevents the development of ventricular remodeling in AMI.