Myocardial Ischemia In Rats Research Articles

Anti-apoptotic effect of microRNA-30b in early phase of rat myocardial ischemia-reperfusion injury model.

This study aimed to investigate the effect of microRNA-30b (miR-30b) in rat myocardial ischemic-reperfusion (I/R) injury model. We randomly divided Sprague-Dawley (SD) rats (n = 80) into five groups: 1) control group; 2) miR-30b group; 3) sham-operated group; 4) I/R group, and 5) I/R+miR-30b group. Real-time quantitative polymerase chain reaction, immunohistochemical staining and Western blot analysis were conducted. TUNEL assay was employed for testing cardiomyocyte apoptosis. Our results showed that miR-30b levels were down-regulated in I/R group and I/R + miR-30b group compared with sham-operated group (both P < 0.05). However, miR-30b level in I/R + miR-30b group was higher than I/R group (P < 0.05). Markedly, the apoptotic rate in I/R group showed highest in I/R group (P < 0.05). Additionally, the results illustrated that protein levels of Bcl-2, Bax, and caspase-3 were at higher levels in ischemic regions in I/R group, comparing to sham-operated group (all P < 0.05), while Bcl-2/Bax was reduced (P < 0.05). Bcl-2 level and Bcl-2/Bax were obviously increased in I/R + miR-30b group by comparison with I/R group, and expression levels of Bax and caspase-3 were down-regulated (all P < 0.05). We also found that in I/R + miR-30b group, KRAS level was apparently lower and p-AKT level was higher by comparing with I/R group (both P < 0.05). Our study indicated that miR-30b overexpression had anti-apoptotic effect on early phase of rat myocardial ischemia injury model through targeting KRAS and activating the Ras/Akt pathway.

Effect of CXCR4 pretreated with ultrasound-exposed microbubbles on accelerating homing of bone marrow mesenchymal stem cells to ischemic myocardium in AMI rats.

To investigate the role and potential mechanism of CXCR4 in promoting targeted homing of bone marrow mesenchymal stem cells (BMSCs) with ultrasound-exposed microbubbles (UM) pretreatment. Third generation BMSCs were divided into four groups control group, ultrasound (US) group, UM group and ultrasound-exposed microbubbles plus catalase group. RT-PCR and western blot were performed to determine the levels of CXCR4 mRNA transcription and protein expression, respectively. Third generation BMSCs were labeled with Fluo-α/AM and divided into three groups: control group, US group and UM group, and fluorescence intensities in the cells were observed immediately, 5min and 15min after intervention under fluorescence microscope. The calcium iron levels in the cells were analyzed. BMSCs were divided into five group: group A without calcium in the medium, group B, group C, group D and group E containing calcium chloride with concentration of l mol, 2mol, 4mol, anti-calcium-sensing receptor antibody, respectively. RT-PCR and western blot were performed to determine the levels of CXCR4 mRNA transcription and proteins expression of the third generation BMSCs of each group, respectively. The levels of CXCR4 mRNA transcription and protein expression between US group and control group had no statistically significant difference (P>0.05) shown by RT-PCR and western blot; the transcription level in the UM group was significantly higher than that in US group and control group (P<0.05); and in the ultrasound-exposed microbubbles plus catalase group, the transcription level was much lower than that in UM group. Fluorescence intensify in the cells of US group had no significant difference compared with that in the cells of the control group (P>0.05), which in the cells of UM group was significantly higher than that in the cells of both US group and control group (P<0.05). Compared to group A, expressions of CXCR4 of group B to D were significantly increased in concentration-dependent manner showed by RT-PCR and western blot (P<0.05). Compared to group C, expressions of CXCR4 of group E were significantly decreased (P<0.05). UM can promote the influx of calcium in BMSCs and increase mRNA transcription and protein expression of CXCR4. The latter may partly be caused by influx of calcium.

Injectable shear-thinning hydrogels used to deliver endothelial progenitor cells, enhance cell engraftment, and improve ischemic myocardium

ObjectivesThe clinical translation of cell-based therapies for ischemic heart disease has been limited because of low cell retention (<1%) within, and poor targeting to, ischemic myocardium. To address these issues, we developed an injectable hyaluronic acid (HA) shear-thinning hydrogel (STG) and endothelial progenitor cell (EPC) construct (STG-EPC). The STG assembles as a result of interactions of adamantine- and β-cyclodextrin–modified HA. It is shear-thinning to permit delivery via a syringe, and self-heals upon injection within the ischemic myocardium. This directed therapy to the ischemic myocardial border zone enables direct cell delivery to address adverse remodeling after myocardial infarction. We hypothesize that this system will enhance vasculogenesis to improve myocardial stabilization in the context of a clinically translatable therapy. MethodsEndothelial progenitor cells (DiLDL+ VEGFR2+ CD34+) were harvested from adult male rats, cultured, and suspended in the STG. In vitro viability was quantified using a live-dead stain of EPCs. The STG-EPC constructs were injected at the border zone of ischemic rat myocardium after acute myocardial infarction (left anterior descending coronary artery ligation). The migration of the enhanced green fluorescent proteins from the construct to ischemic myocardium was analyzed using fluorescent microscopy. Vasculogenesis, myocardial remodeling, and hemodynamic function were analyzed in 4 groups: control (phosphate buffered saline injection); intramyocardial injection of EPCs alone; injection of the STG alone; and treatment with the STG-EPC construct. Hemodynamics and ventricular geometry were quantified using echocardiography and Doppler flow analysis. ResultsEndothelial progenitor cells demonstrated viability within the STG. A marked increase in EPC engraftment was observed 1-week postinjection within the treated myocardium with gel delivery, compared with EPC injection alone (17.2 ± 0.8 cells per high power field (HPF) vs 3.5 cells ± 1.3 cells per HPF, P = .0002). A statistically significant increase in vasculogenesis was noted with the STG-EPC construct (15.3 ± 5.8 vessels per HPF), compared with the control (P < .0001), EPC (P < .0001), and STG (P < .0001) groups. Statistically significant improvements in ventricular function, scar fraction, and geometry were noted after STG-EPC treatment compared with the control. ConclusionsA novel injectable shear-thinning HA hydrogel seeded with EPCs enhanced cell retention and vasculogenesis after delivery to ischemic myocardium. This therapy limited adverse myocardial remodeling while preserving contractility.

Spironolactone Regulates HCN Protein Expression Through Micro-RNA-1 in Rats With Myocardial Infarction.

:Emerging evidence has shown that aldosterone blockers reduced the incidence of ventricular arrhythmias in patients with myocardial infarction (MI). However, the mechanism remains unknown. In this study, we investigated the mechanism by which spironolactone, a classic aldosterone blocker, regulates hyperpolarization-activated cyclic nucleotide-gated channel (HCN) protein expression in ischemic rat myocardium after MI. Eighteen rats surviving 24 hours after MI were randomly assigned into 3 groups: MI, spironolactone, and spironolactone + antagomir-1. Six sham-operated rats had a suture loosely tied around the left coronary artery, without ligation. The border zone of the myocardial infarct was collected from each rat at 1 week after MI. HCN2 and HCN4 protein and messenger RNA (mRNA) level were measured in addition to miRNA-1 levels. Spironolactone significantly increased miRNA-1 levels and downregulated HCN2 and HCN4 protein and mRNA levels. miRNA-1 suppression with antagomir-1 increased HCN2 and HCN4 protein levels; however, HCN2 and HCN4 mRNA levels were not affected. These results suggested that spironolactone could increase miRNA-1 expression in ischemic rat myocardium after MI and that the upregulation of miRNA-1 expression partially contributed to the posttranscriptional repression of HCN protein expression, which may contribute to the effect of spironolactone to reduce the incidence of MI-associated ventricular arrhythmias.

Protective effects of tanshinone IIA on myocardial ischemia reperfusion injury by reducing oxidative stress, HMGB1 expression, and inflammatory reaction

Context: Although there were reports on the protective functions of tanshinone IIA (TSA) on rat myocardial ischemia, the exerting mechanism has not been completely clarified.Objective: An attempt was made to further verify the protective effect of TSA on myocardial ischemia reperfusion injury and elucidate its underlying mechanism.Materials and methods: The rats were given TSA (10, 20, and 40 mg/kg bw per day) in intraperitoneal injection for 15 d. Rami anterior descending branch of coronary artery was ligated for 30 min and then re-perfused for 120 min to establish a reperfusion model. Effects of TSA on the infarct area, creatine kinase (CK), aspartate aminotransferase (AST), high mobility group box B1 protein (HMGB1), and inflammation and oxidation were investigated.Results: Compared with those in the IR group, infarct size percentages of rats’ myocardium in L-TSA, M-TSA, and H-TSA groups were reduced by 1.21, 4.26, and 12.50%, respectively, CK activities by 7.4, 11.2, and 12.5%, respectively, and AST activities also declined (p < 0.05). Furthermore, compared with those in the IR group, SOD and GSH-Px activities increased, and MDA, TNF-α, IL-6, and iNOS levels decreased in L-TSA, M-TSA, and H-TSA groups (p < 0.05). Meanwhile, compared with those in the IR group, HMGB1 expressions in L-TSA, M-TSA, and H-TSA groups were lowered by 21.9, 32.4, and 35.6%, respectively.Discussion and conclusion: The protective function of TSA on myocardial ischemia reperfusion injury may be possibly exerted by inhibiting the increase of ROS caused by the reperfusion to attenuate the expression of HMGB1 and inhibit inflammation.

Urinary metabolomic strategy to evaluate Compound Danshen Dripping Pills for myocardial ischaemia in rats

Compound Danshen Dripping Pill (CDDP) has been used for the treatment of coronary heart disease for decades. We aimed to increase the understanding of the mechanisms by evaluating the urinary metabolomics of CDDP using Gas Chromatography-Mass Spectrometer (GC-MS) in a myocardial ischaemia (MI) rat model. One hundred Sprague-Dawley rats were divided into Con (normal saline and no surgery), Con+ (107mg/kgd CDDP solution and no surgery), Sham (normal saline and surgery without aorta ligation), Mod (normal saline and surgery with aorta ligation), and Mod+ (107mg/kgd CDDP solution and surgery with aorta ligation) groups. Urine samples on days 0, 3, 14, and 28 were tested using GC-MS and analyzed with PCA and partial least squares-discriminant analysis models. In the Mod group, creatine kinase and malondialdehyde levels were higher, and superoxide-dismutase levels were lower; the same variables normalized in the Mod+ group. CDDP resulted in improvement in the Mod+ group, as indicated by the reduced necrosis in the myocardial tissue. A total of 36 metabolites were identified in the urine samples, and 8 metabolites (malate, succinate, creatinine, methionine, cysteine, serine, phenylalanine, and tyrosine) were increased remarkably and recovered to normal levels after treatment with CDDP. Differentially expressed metabolites implied that energy, amino acid, fatty acid, and polyol metabolism might be disrupted by MI and reversed by CDDP. Urinary metabolomics provide a dynamic monitoring approach that highlights interference by MI and the therapeutic effects of CDDP on MI in rats throughout the recovery process.

A chitosan-graft-PEI-eprosartan conjugate for cardiomyocyte-targeted VEGF plasmid delivery in myocardial ischemia gene therapy

A multifunctional copolymer conjugate chitosan-graft-polyethyleneimine-eprosartan (CPE) containing low-molecular-weight chitosan backbone and polyethyleneimine (PEI) arms with eprosartan (ES) conjugated via an amide bond was fabricated as a cardiomyocyte-targeted vascular endothelial growth factor (VEGF) plasmid delivery nanovector. Here, ES was utilised to specifically bind to overexpressed angiotensin II type 1 receptor (AT1R) of cardiomyocytes and strengthen endosomal buffering capacity of CPE. The self-assembled CPE/VEGF complexes exhibited desirable and homogenous particle size, moderate positive charges, advanced gene condensation and protection profiles in vitro. Flow cytometry and ELISA analyses confirmed that ES-targeted function and ES-enhanced buffering capacity induced specific cellular uptake and high transfection of CPE/VEGF complexes in AT1R-overexpressed H9C2 cells. In vivo investigation on myocardial ischemia rat model revealed that CPE/VEGF complexes possessed strong therapeutic effect. These findings suggested that CPE could be an ideal cardiomyocyte-targeted nanovector for effective transfer of VEGF plasmid, and a multifunctional CPE/VEGF delivery system with cardiomyocyte-specific targetability, enhanced endosomal buffering capacity and strong therapeutic effect might be a new promising strategy for effective myocardial ischemia gene therapy.

Protein and RNA expression of acid-sensing ion channels 2 and 3 in myocardial ischemia rats induced by isoproterenol

ObjectiveTo investigate the impact of electro-acupuncture at the Neiguan (PC 6) acupoint on protein and RNA expression of acid-sensing ion channel 2 (ASIC2) and ASIC3 in myocardial ischemia rats. MethodsFifty male Sprague-Dawley rats were used, weighing (230 ± 50) g. The rats were randomized into a normal group A, model group B, Neiguan (PC 6) group C, Lieque (LU 7) group D, and A-shi points group E. There were 10 rats in each group. Rats were continuously administered 85 mg/kg intravenous isoproterenol daily to establish the model. Successfully modeled rats in groups C, D, and E were given electro-acupuncture treatment. Each group of rats was sacrificed with chloral hydrate (1 mL/100 g) intraperitoneal injection. The left ventricular myocardium was extracted and placed at − 70 °C until use. Western blot analysis and real-time PCR were performed to assay protein and RNA expressions of ASIC2 and ASIC3, respectively. Fold changes in RNA expression were quantified with the 2∧(− ΔΔCt) method. Blood samples were drawn from the aorta abdominalis and tested for creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) levels using enzyme-linked immunosorbent assay. ResultsMyocardial ischemia rats given electro-acupuncture at the Neiguan (PC 6) acupoint had significantly lower protein and RNA expression of ASIC2 and ASIC3, and CK-MB and LDH levels, compared with model rats (P < 0.01). ConclusionElectro-acupuncture at the Neiguan (PC 6) acupoint can not only decrease the protein and RNA expression of ASIC2 and ASIC3, but also inhibit the opening of ASICs and reduce the cardiomyocyte damage in myocardial ischemia rats.

Mechanisms of isoproterenol-induced cardiac mitochondrial damage: protective actions of melatonin.

Mitochondrial dysfunction due to oxidative damage is the key feature of several diseases. We have earlier reported mitochondrial damage resulting from the generation of oxidative stress as a major pathophysiological effect of isoproterenol (ISO)-induced myocardial ischemia in rats. That melatonin is an antioxidant that ameliorates oxidative stress in experimental animals as well as in humans is well established. We previously demonstrated that melatonin provides cardioprotection against ISO-induced myocardial injury as a result of its antioxidant properties. The mechanism of ISO-induced cardiac mitochondrial damage and protection by melatonin, however, remains to be elucidated in vitro. In this study, we provide evidence that ISO causes dysfunction of isolated goat heart mitochondria. Incubation of cardiac mitochondria with increasing concentrations of ISO decreased mitochondrial succinate dehydrogenase (SDH) activity, which plays a pivotal role in mitochondrial bioenergetics, as well as altered the activities of other key enzymes of the Kreb's cycle and the respiratory chain. Co-incubation of ISO-challenged mitochondria with melatonin prevented the alterations in enzyme activity. That these changes in mitochondrial energy metabolism were due to the perpetration of oxidative stress by ISO was evident from the increased levels of lipid peroxidation and decreased reduced glutathione/oxidized glutathione ratio. ISO-induced oxidative stress also altered mitochondrial redox potential and brought about changes in the activity of the antioxidant enzymes manganese superoxide dismutase and glutathione peroxidase, eventually leading to alterations in total ATPase activity and membrane potential. Melatonin ameliorated these changes likely through its antioxidant abilities suggesting a possible mechanism of cardioprotection by this indole against ISO-induced myocardial injury.

Danqi Pill regulates lipid metabolism disorder induced by myocardial ischemia through FATP-CPTI pathway.

BackgroundDanqi Pill (DQP), which contains Chinese herbs Salvia miltiorrhiza Bunge and Panax notoginseng, is widely used in the treatment of myocardial ischemia (MI) in China. Its regulatory effects on MI-associated lipid metabolism disorders haven’t been comprehensively studied so far. We aimed to systematically investigate the regulatory mechanism of DQP on myocardial ischemia-induced lipid metabolism disorders.MethodsMyocardial ischemia rat model was induced by left anterior descending coronary artery ligation. The rat models were divided into three groups: model group with administration of normal saline, study group with administration of DanQi aqueous solution (1.5 mg/kg) and positive-control group with administration of pravastatin aqueous solution (1.2 mg/kg). In addition, another sham-operated group was set as negative control. At 28 days after treatment, cardiac function and degree of lipid metabolism disorders in rats of different groups were measured.ResultsPlasma lipid disorders were induced by myocardial ischemia, with manifestation of up-regulation of triglyceride (TG), low density lipoprotein (LDL), Apolipoprotein B (Apo-B) and 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGCR). DQP could down-regulate the levels of TG, LDL, Apo-B and HMGCR. The Lipid transport pathway, fatty acids transport protein (FATP) and Carnitine palmitoyltransferase I (CPTI) were down-regulated in model group. DQP could improve plasma lipid metabolism by up-regulating this lipid transport pathway. The transcription factors peroxisome proliferator-activated receptor α (PPARα) and retinoid X receptors (RXRs), which regulate lipid metabolism, were also up-regulated by DQP. Furthermore, DQP was able to improve heart function and up-regulate ejection fraction (EF) by increasing the cardiac diastolic volume.ConclusionsOur study reveals that DQP would be an ideal alternative drug for the treatment of dyslipidemia which is induced by myocardial ischemia.

A computational and functional study elicits the ameliorating effect of the Chinese herbal formula Huo Luo Xiao Ling Dan on experimental ischemia-induced myocardial injury in rats via inhibition of apoptosis.

Ischemic heart disease (IHD) is the leading cause of death worldwide and remains a major life-threatening factor in humans. Apoptosis has been implicated in the pathogenesis of IHD. The Chinese herbal formula Huo Luo Xiao Ling Dan (HLXLD), one of the commonly used Chinese herbal formulas, consists of Salviae miltiorrhizae, Angelica sinensis, Gummi olibanum, and Commiphora myrrha, with a wide spectrum of pharmacological activity. However, the mechanism of action and molecular targets of HLXLD in the treatment of IHD are unclear. This study aimed to computationally predict the molecular interactions between the major active components of HLXLD and key regulators of apoptosis and then examine the effect of HLXLD on coronary artery ligation-induced acute myocardial ischemia in rats. The molecular interactions between the major active components of HLXLD, including ferulic acid, ligustilide, succinic acid, vanillic acid, tanshinone IIA, tanshinone IIB, danshensu, salvianolic acid A, salvianolic acid C, protocatechuic aldehyde, and β-boswellic acid and human protein molecules including B cell lymphoma-extra large (Bcl-xl), B cell lymphoma 2 antagonist/killer 1 (Bak1), B cell lymphoma 2 (Bcl-2), procaspase 3, and caspase 9 with regard to hydrogen bond formation, charge interaction, and π-π stacking using Discovery Studio® program 3.1. The 12 HLXLD components were predicted by ADMET (absorption, distribution, metabolism, excretion and toxicity) Predictor to have favorable pharmacokinetic and low hepatotoxicity profiles. The acute myocardial ischemia was established by surgical ligation of the left anterior descending coronary artery. The rats were divided into a sham operative group, a model group, a positive control group treated with 0.2 mg/kg isosorbide mononitrate, and groups treated with 2.7, 5.4, or 10.8 g/kg HLXLD. The results showed that administration of HLXLD increased mean arterial pressure, left ventricular systolic pressure, heart rate, and maximal rate of rise/descent of left ventricular pressure levels. Administration of HLXLD significantly ameliorated coronary artery ligation-induced tissue damage in the left ventricle, with restored arrangement of myocardial fibers and recovered myoplasm in rats. Furthermore, HLXLD markedly increased the expression level of Bcl-2 but decreased the level of cleaved caspase 3. Taken together, administration of HLXLD attenuated acute myocardial ischemia-induced damage in cardiomyocytes and inhibited apoptotic death of cardiomyocytes, thereby exerting a cardioprotective effect in rats with IHD. These findings suggest that HLXLD may represent a promising herbal formula for the treatment of cardiovascular disease by counteracting apoptotic cell death via multiple active compounds. More studies are warranted to fully elucidate the mechanisms of action, identify the therapeutic targets, and validate the efficacy and safety of HLXLD in the treatment of IHD.

Evaluation on contribution rate of each component total salvianolic acids and characterization of apparent oil/water partition coefficient

The difference between three representative components of total salvianolic acids in pharmacodynamic activity were compared by three different pharmacological experiments: HUVECs oxidative damage experiment, 4 items of blood coagulation in vitro experiment in rabbits and experimental myocardial ischemia in rats. And the effects of contribution rate of each component were calculated by multi index comprehensive evaluation method based on CRITIC weights. The contribution rates of salvianolic acid B, rosmarinic acid and Danshensu were 28.85%, 30.11%, 41.04%. Apparent oil/water partition coefficient of each representative components of total salvianolic acids in n-octyl alcohol-buffer was tested and the total salvianolic acid components were characterized based on a combination of the approach of self-defined weighting coefficient with effects of contribution rate. Apparent oil/water partition coefficient of total salvianolic acids was 0.32, 1.06, 0.89, 0.98, 0.90, 0.13, 0.02, 0.20, 0.56 when in octanol-water/pH 1.2 dilute hydrochloric acid solution/ pH 2.0, 2.5, 5.0, 5.8, 6.8, 7.4, 7.8 phosphate buffer solution. It provides a certain reference for the characterization of components.

Effect of hydroxy safflower yellow A on myocardial apoptosis after acute myocardial infarction in rats.

This study aimed to investigate the effect of hydroxy safflower yellow A (HSYA) on myocardial apoptosis after acute myocardial infarction (AMI) in rats. We randomly divided 170 male Wistar rats into 6 groups (N = 23): normal control, sham, control, SY (90 mg/kg), HSYA high-dose (HSYA-H, 40 mg/kg), and HSYA low-dose groups (HSYA-L, 20 mg/kg). Myocardial ischemic injury was induced by ligating the anterior descending coronary artery, and the degree of myocardial ischemia was evaluated using electrocardiography and nitroblue tetrazolium staining. Bax and Bcl-2 expressions in the ischemic myocardium were determined using immunohistochemical analysis. Peroxisome proliferator-activated receptor-γ (PPAR-γ) expression in the myocardium of rats with AMI was determined using reverse transcription-polymerase chain reaction. Compared to rats in the control group, those in the HYSA-H, HSYA-L, and SY groups showed a decrease in the elevated ST segments and an increase in the infarct size. The rats in the drug-treated groups showed a significantly lower percentage of Bax-positive cells and a significantly higher percentage of Bcl-2-positive cells than those in the control group (P < 0.05). Moreover, mRNA expression of PPAR-γ in the ischemic myocardium of rats in the SY, HSYA-L, and HSYA-H groups was significantly lower than that in the control group (P < 0.05). Thus, HSYA and SY can attenuate myocardial ischemia in rats, possibly by increasing the level of Bcl-2/Bax, and PPAR-γ may be not a necessary link in this process.

Developing a Nanocarrier for Targeted Delivery of Cardio-Protective Agents

Background: Ischemic heart disease (IHD) is a leading cause of death worldwide. Pre-clinical drugs for IHD have largely failed in human clinical trials, highlighting the importance of developing vehicles to selectively deliver optimal therapeutic agents to the ischemic myocardium. Hydrophobins are fungal proteins that self-assemble into robust amphipathic monolayers that can encapsulate and solubilise hydrophobic drugs.Aim: Develop the non-immunogenic hydrophobin RodA as a targeted-drug delivery vehicle.Methods: Three recombinant RodA variants were prepared: RodA-MY, engineered to contain the peptide CSTSMLKAC that selectively binds to ischemic myocardium and RodA with or without a FITC-conjugate. RodA monomers and polymers were produced with and without the targeting sequence. All variants were tested for the ability of self-assemble into fibrillar (nanocarrier) structures. Monomeric and polymeric RodA variants were tested for cardiotoxicity using cardiomyocyte-like H9c2 cells. Targeting ability was tested using ischemia-challenged H9c2 cells and ischemic myocardium from a rat model of myocardial ischemia reperfusion.Results and conclusions: Hydrophobins were well-tolerated by cultured H9c2 cells. Exposure of cells to both monomeric RodA or RodA polymers (50-100µg/mL) had no effect on cell viability or cell cycle profile. Immunocytochemistry demonstrated unaltered cell surface receptor distribution and activity as judged by binding and uptake of FITC-labelled transferrin. Consistent with previous data, the levels of the pro-inflammatory gene nFkB were unchanged suggesting that hydrophobins did not elicit inflammation in cardiac cells. Although, RodA-MY-nanocarrier has advantages over the native RodA-nanocarrier in targeting both normoxic and injured H9C2 cells, pilot work has revealed that RodA-MY-nanocarrier preferentially binds ex vivo to ischemic rat myocardium. Together the data indicate that hydrophobin-based polymers may act as drug-delivery vehicles for myocardial ischemic pathologies. Future work: studying the vehicles targeted ability of passing the vasculature barriers.

UHPLC-LTQ-Orbitrap MS combined with spike-in method for plasma metabonomics analysis of acute myocardial ischemia rats and pretreatment effect of Danqi Tongmai tablet.

Undoubtedly, metabonomics can reveal the comprehensive efficacies of traditional Chinese medicine (TCM) formulae and its complex mechanism at the molecular biological level. In this study, an attempt was made to address the pretreatment effect of a TCM formula. In this case, as a critical point, we should first know how to really reflect the various endogenous metabolites in a disease status before a TCM formula is employed in a therapeutic procedure. Here, we explored an approach that combined high resolution LTQ-Orbitrap mass spectrometry with a spike-in method to characterize endogenous metabolites in acute myocardial ischemia (AMI) rats. As a result, 19 potential biomarkers in rat plasma were identified and 10 related disturbed pathways were perturbed in the early stages of AMI development. Subsequently, the metabonomics method was applied to investigate the pretreatment effect of the TCM formula named the Danqi Tongmai tablet (DQTM). The results revealed that the DQTM pretreatment could reduce the AMI injury and partially regulate the perturbed TCA cycle and amino and nucleotide metabolism, which were presumable related to energy metabolism and myocardial cells apoptosis/necrosis. In conclusion, UHPLC-LTQ-Orbitrap MS combined with a spike-in method were successfully applied to the metabonomics analysis of DQTM, which demonstrated that not only a comprehensive metabolic profile in the early stages of AMI development was achieved, but also that the underlying holistic efficacies were assessed and it was helpful to understand the possible mechanism of pretreatment with DQTM.

Protective effects of acacetin isolated from Ziziphora clinopodioides Lam. (Xintahua) on neonatal rat cardiomyocytes

BackgroundThe total flavonoids from ethanol extract of the aerial part of Ziziphora clinopodioides Lam. (Lamlaceae) (Xintahua) showed protective activities against rat acute myocardial ischemia in rats. This study aims to isolate acacetin, a flavonoid, from the aerial part of Z. clinopodioides, to develop an HPLC method for its detection, and to evaluate its protective effects on neonatal rat cardiomyocytes.MethodsSephadex LH-20 silicagel and pillar layer chromatography silica gel were applied for the isolation and purification of acacetin and its structure was elucidated on the basis of 1H and 13C NMR spectroscopy. The content of acacetin in Z. clinopodioides collected from three different origins was determined by HPLC. The neonatal rat cardiomyocytes were isolated and cultured in vitro to establish a hypoxia/reoxygenation injury model. The viability of cardiomyocytes was measured by the MTT method. Changes of malondialdehyde (MDA) content in the medium were also determined.ResultsThe acacetin content in various batches of Z. clinopodioides ranged from 45.50 to 47.41 μg/g. Acacetin of 25, 10, 5 μg/mL significantly decreased the MDA content in a model of hypoxia/reoxygenation injury (P < 0.001, P < 0.001 and P = 0.033, respectively).ConclusionsAcacetin protects neonatal cardiomyocytes from the damage induced by hypoxia/reoxygenation stress through reduction of lipid peroxidation and enhancement of the antioxidant activity.

Effect of aminoguanidine on cell apoptosis induced by acute myocardial ischemia in rats

Objective To evaluate the effect of aminoguanidine on cell apoptosis induced by acute myocardial ischemia in rats.Methods Thirty adult male Sprague-Dawley rats,weighing 250-290 g,were randomly divided into 3 groups (n =10 each):sham operation group (group S),myocardial ischemia group (group Ⅰ),and aminoguanidine group (group AG).The model of acute myocardial ischemia was established by ligating the left anterior descending branch of the coronary artery of rats anesthetized with chloral hydrate.Aminoguanidine 100 mg/kg was intraperitoneally administrated at 6 h of ischemia in AG group,while the equal volume of normal saline was given instead of aminoguanidine in group Ⅰ.The chest was opened at 3 h after aminoguanidine administration and hearts were quickly removed for detection of apoptosis in cardiomyocytes (by TUNEL) and expression of Bcl-2 and Bax in cardiomyocytes (by immuno-histochemistry),and for microscopic examination with light microscope.Apoptotic rate was calculated.Results Compared with group S,the apoptotic rate was significantly increased,the expression of Bax was up-regulated,and the expression of Bcl-2 was downregulated and the ratio of Bcl-2/Bax was decreased in group Ⅰ.Compared with group Ⅰ,the apoptotic rate was significantly decreased,the expression of Bax was down-regulated,and the expression of Bcl-2 was up-regulated,and the ratio of Bcl-2/Bax was increased in group AG.The pathological changes of myocardial cells were significantly attenuated in AG group as compared with group Ⅰ.Conclusion Amionguanidine can inhibit apoptosis in cardiomyocytes and is helpful in mitigating injury induced by acute myocardial ischemia in rats. Key words: Guanidines; Myocardical ischemia ; Apopotosis

The beneficial effect of electro-acupuncture given at PC6 (Neiguan-point) by the increase in cardiac transient outward K+ current channel which depends on the gene and protein expressions in artificially induced myocardial ischemia rats.

This study explored the mechanism of electro-acupuncture (EA) at PC6 to improve the heart function by regulating the cardiac transient outward potassium current (= Ito) channel in myocardial ischemia (MI). Kv1.4 is the main component of the slow Ito (Ito.s) channel. Kv4.2 and Kv4.3 are the main components of the fast Ito (Ito.f) channel. KChIP2 is a compound protein of Ito channel. In this experiment, MI was induced by injecting isoproterenol in rats, and the gene and protein expressions of Kv1.4, Kv4.2, Kv4.3 and KChIP2 were lower [The gene and protein decreased (42.8 ± 4.3)% and (37.2 ± 4.7)% respectively.] than the control rats significantly (p < 0.05). After MI induction, the MI rats were divided into three groups, including PC6, LU7 (Lieque-point) and Non-acupoint group which were acupunctured at once a day for 7 days respectively. After EA at PC6 and LU7, the gene and protein expressions showed obvious increased [EA at PC6: the gene and protein increased (50.24 ± 5.4)% and (39.14 ± 5.3)% respectively. EA at LU7: the gene and protein increased (24.6 ± 2.8)% and (22.04 ± 5.5)% respectively] and they are all higher than MI rats significantly (p < 0.05). EA at PC6 and LU7 showed the same variation, and the effects of EA at PC6 and LU7 were better than Non-acupuncture-point (p < 0.05). The effects of EA at PC6 were significantly better [The gene and protein increased (19.74 ± 2.7)% and (14.14 ± 4.4)% respectively] than LU7 (p < 0.05). PC6 is an acupoint of the pericardium meridian, and the pericardium meridian which corresponds to adrenal gland according to Omura Y's research, can affect the heart function directly LU7 belong to the lung meridian, and the lung plays an important factor in blood circulation according to TCM. So PC6 is more effective than LU7 on heart function regulation. The results suggested that PC6 showed the target effect of meridian specificity on regulating the Kv channel in MI.

Effects of prostaglandin E1 on nitric oxide and oxygen dynamics during rat myocardial ischemia–reperfusion utilizing sol–gel derived microsensors

Prostaglandin E1 (Alprostadil®, PGE1) is often used for patients with critical limb ischemia due to its vasodilator and anti-platelet effects. Although PGE1 is considered to have a cardioprotective effect against myocardial ischemia–reperfusion (IR) injury, its mechanism and correlation with nitric oxide (NO) for cardioprotection are not clear. In this study, we used sol–gel-modified electrochemical NO and O2 microsensors to simultaneously measure the changes in NO level and oxygen tension (pO2) in the myocardium during myocardial IR that were induced by PGE1 pretreatment. Rat hearts were randomly divided into two groups: a control group (n=5) and a PGE1 group (n=5, with PGE1 pretreatment). Myocardium that underwent PGE1 pretreatment (26.0±13.1%, p<0.001) demonstrated more highly reduced endogenous NO production during the ischemic period than that of the control group (86.7±18.5%). After the onset of reperfusion, %NO level in the PGE1 group increased to a maximum of 82.0±6.4 but did not exceed the pre-ischemic basal NO level. However, in the control group, the maximum %NO response (164.9±41.0) on reperfusion was approximately double that of the PGE1 group (p<0.01, n=5). The restoration value of pO2 in the PGE1 group significantly increased and was restored to pre-ischemic level (82.2±21.3%); however, the pO2 of the control group did not increase throughout the reperfusion period (12.3±7.9%, p<0.001). As a result, we suggest that the cardioprotective effect of PGE1 might be attributed to a reduction in excessive NO production during reperfusion. Therefore, simultaneous and real-time monitoring of NO and pO2 dynamics in the myocardium would be effective in evaluating the effects of therapeutic treatments such as drug administration. Also, the reduction in excessive NO release during the early reperfusion period might be helpful as a therapeutic strategy to protect the myocardium from IR injury.

Tissue-engineered, hydrogel-based endothelial progenitor cell therapy robustly revascularizes ischemic myocardium and preserves ventricular function

Cell-based angiogenic therapy for ischemic heart failure has had limited clinical impact, likely related to low cell retention (<1%) and dispersion. We developed a novel, tissue-engineered, hydrogel-based cell-delivery strategy to overcome these limitations and provide prolonged regional retention of myocardial endothelial progenitor cells at high cell dosage. Endothelial progenitor cells were isolated from Wistar rats and encapsulated in fibrin gels. In vitro viability was quantified using a fluorescent live-dead stain of transgenic enhanced green fluorescent protein(+) endothelial progenitor cells. Endothelial progenitor cell-laden constructs were implanted onto ischemic rat myocardium in a model of acute myocardial infarction (left anterior descending ligation) for 4 weeks. Intramyocardial cell injection (2 × 10(6) endothelial progenitor cells), empty fibrin, and isolated left anterior descending ligation groups served as controls. Hemodynamics were quantified using echocardiography, Doppler flow analysis, and intraventricular pressure-volume analysis. Vasculogenesis and ventricular geometry were quantified. Endothelial progenitor cell migration was analyzed by using endothelial progenitor cells from transgenic enhanced green fluorescent protein(+) rodents. Endothelial progenitor cells demonstrated an overall 88.7% viability for all matrix and cell conditions investigated after 48 hours. Histologic assessment of 1-week implants demonstrated significant migration of transgenic enhanced green fluorescent protein(+) endothelial progenitor cells from the fibrin matrix to the infarcted myocardium compared with intramyocardial cell injection (28 ± 12.3 cells/high power field vs 2.4 ± 2.1 cells/high power field, P = .0001). We also observed a marked increase in vasculogenesis at the implant site. Significant improvements in ventricular hemodynamics and geometry were present after endothelial progenitor cell-hydrogel therapy compared with control. We present a tissue-engineered, hydrogel-based endothelial progenitor cell-mediated therapy to enhance cell delivery, cell retention, vasculogenesis, and preservation of myocardial structure and function.