Ischemic Myocardial Cells Research Articles

Effects of intracoronary supersaturated oxygen infusion on the time course of myocardial blood flow and extent of microvascular obstruction in an ischemia/reperfusion preclinical swine model

Abstract Introduction Despite successful epicardial reperfusion with primary PCI, reactive changes in microvascular function lead to impaired myocardial reperfusion. Intracoronary SuperSaturated Oxygen Therapy (SSO2) is the only approved therapy that improves myocardial salvage in STEMI pts through the delivery of hyperbaric levels of oxygen to viable ischemic vascular and myocardial cells. Several important questions remain unanswered regarding the potential mechanisms of this benefit. Therefore, the purpose of this study is to determine the effects of SSO2 on the time course of myocardial blood flow (MBF) and the extent of microvascular obstruction (MVO) in an ischemia/reperfusion model. Methods 10 swine (32-40 kg) surviving 90-minute balloon-induced anterior STEMI, and 15-min auto reperfusion, were assigned to 120-min of SSO2 (n=5) or control (n=5) with 120-min auto reperfusion. MBF was evaluated at various time points during the experiment by injection of 15–µm microspheres (BioPal, USA) through a catheter placed in the left ventricle. Before euthanasia, 4% thioflavin-S (TS) was injected into the left anterior descending artery. The left ventricle was sectioned into 10-mm thick slices, and the myocardial rings were photographed under UV light to determine areas at risk (TS positive), MVO (lack of TS staining within TS positive area), and remote zones (contiguous areas unstained by TS). MBF was calculated as the total blood flow within the areas at risk normalized to the total flow within the remote zones. Results All animals experienced significant reduction (p = 0.02) in MBF due to balloon occlusion at the 85-minute MI timepoint compared to baseline (Fig). Following the removal of occlusion, reactive hyperemic flows were observed for both groups at the 15-minute auto reperfusion time point (Fig). Starting at the 30-min time point of autoperfusion to 120-min, the MBF significantly decreased (p=0.02) in the controls (Fig). In contrast during these time points, for the SSO2 group the MBF values were numerically higher than the controls, with average MBF for the SSO2 group being significantly higher (p = 0.03) than the controls (1.1 ± 0.18 vs 0.6 ± 0.13, respectively) at the 120-min timepoint (Fig). MVO was found within the areas at risk in all animals, but the extent of MVO in relation to the area at risk was significantly reduced (p < 0.001) in the SSO2 group versus controls (15.2 ± 7.3% vs 41.8% ± 6.2%, respectively). Conclusions Results from this study showed that in a STEMI swine model, SSO2 prevented reduction in MBF during the 120-min reperfusion period, with significantly increased MBF at the end of the experiment compared to controls. This flow improvement translated to a 64% relative reduction in the extent of MVO compared to controls. These results suggest that one of the mechanisms by which SSO2 improves myocardial salvage may be by limiting early microcirculatory alterations set in motion by a cascade of reduced myocardial flow and MVO.Myocardial Blood Flow vs Time

Pharmacological treatment of stable angina pectoris: the place of trimetazidine

According to international epidemiological studies, the total number of reported cases of cardiovascular diseases (CVD) almost doubled from 1990 to 2019, reaching 523 million and the number of deaths from CVD in 2019 increased by more than 1.5 times (18,6 million). Coronary artery disease (CAD) and stroke are the main contributors to these unfavorable trends. The number of registered cases of coronary heart disease in 2019 amounted to 197 million, and the number of deaths caused by coronary artery disease exceeded half of all registered cases of cardiovascular death (9,14 million). Patients with stable angina are the majority of patients with CAD. Despite the existing modern methods of treating angina pectoris, patients with chronic coronary artery disease continue to suffer from anginal pain, which significantly reduces exercise tolerance and worsens their quality of life. In clinical practice, the severity and frequency of angina pectoris in patients remain underestimated by doctors, and drug therapy is not corrected in a timely manner, and the possibilities of combined antianginal therapy are not used. Trimetazidine, as an antianginal drug that acts on the metabolism of ischemic myocardial cells (influence on the ischemic cascade, by reducing cellular acidosis and increasing ATP content), is effective and safety for the treatment of angina pectoris, regardless of the mechanism that caused ischemia as monotherapy and in the combination, primarily with beta-blockers.

Inhibition of coagulation factor XI attenuates inflammation in myocardial ischemia/reperfusion injury

Abstract Background Increasing evidence suggests that FXI is an attractive target for antithrombotic therapy because it reduces thrombosis without increasing bleeding risk. Patients with decreased levels of FXI are at reduced risk of cardiovascular diseases and thromboembolic events. We have already revealed that depletion of FXI inhibits the vascular coagulation-inflammatory circuit in angiotensin II-induced arterial hypertension. However, the effect of FXI depletion on cardiac inflammation and vascular function in ischemia/reperfusion (I/R) injury is unknown. Purpose Using FXI-depleted mice to investigate the role of FXI in inflammation response post cardiac I/R injury. Methods 8–12 weeks old C57BL/6J male mice were injected intraperitoneally with FXI antisense oligonucleotide (FXI ASO) or scrambled controls for a period of 2 weeks. Then we temporarily ligated the left anterior descending (LAD) for 45 minutes to induce myocardial ischemia, followed by the reperfusion for 72 hours (ischemia/reperfusion injury, IRI). Myocardial inflammation and vascular function were analyzed by flow cytometry, real-time PCR, vascular relaxation studies from isolated aortic segment in organ chamber and chemiluminescence photon counting of oxidative burst in whole blood. Results FXI ASO treatment reduced hepatic FXI mRNA levels and prolonged activated partial thromboplastin time. Compared to scrambled ASO injected mice, oxidative burst from whole blood and endothelial dysfunction were decreased in FXI ASO injected mice with IRI. Compared to control groups, depletion of FXI attenuated cardiac infiltration of CD45+CD11b+ cells, especially LyG-LyChigh monocytes and LyG+ neutrophils at day 3 in I/R injury. Furthermore, the expression levels of adhesion molecules and expression of pro-inflammatory cytokines, such as Vcam-1, CCL2, IL-6 and IL-1b, in ischemic myocardium were significantly decreased in the FXI depleted mice of I/R injury. Conclusion Our results suggest that Inhibition of FXI leads to reduction of vascular dysfunction and ROS production in I/R injury, as well as attenuation of the inflammatory response and the influx of inflammatory cells in ischemic myocardium. This indicates that FXI could be a potential target for further treatment in cardiac IRI. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): TICARDIO project has received funding from the European Union's Horizon 2020 research and Innovation programme under the Marie Skłodowska-Curie grant agreement No 813409.

Protective Effect of Ginsenosides Rg1 on Ischemic Injury of Cardiomyocytes After Acute Myocardial Infarction.

Acute myocardial infarction (AMI) leads to anoxia and ischemia of cardiomyocytes, followed by apoptosis. This study investigated the protective effect of ginsenoside Rg1 (Rg1) on myocardial ischemia injury in rats with AMI. Rats were randomly divided into five groups: group A (blank control group), group B (hypoxia/reoxygenation group), group C (hypoxia/reoxygenation + 10mg/L Rg1), group D (hypoxia/reoxygenation + 20mg/L Rg1) and group E (hypoxia/reoxygenation + 40mg/L Rg1). The survival rate, apoptosis rate, expression of cyclin-dependent kinase 4 (CDK4), fibroblast growth factor 9 (FGF9), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), microvessel density and myocardial infarction area of rats in each group were compared. The expressions of CDK4 and FGF9, the contents of SOD and GSH-Px in groups C, D and E injected with Rg1 were significantly promoted compared to group B without Rg1 injection (P < 0.05). The survival rate of myocardial cells was significantly increased while the apoptosis rate was significantly decreased in group C, D, E compared to group B (P < 0.05). On the 3rd, 7th and 10th day following Rg1 treatment, the infarct area of E group was significantly decreased in three groups C, D, E, and the microvessel density of infarct area was significantly increased compared with group B (P < 0.05). So, Rg1 can improve the survival rate of myocardial cells, reduce the apoptosis rate and the area of myocardial infarction, and increase the microvessel density of infarct area, thus playing a protective role in ischemic myocardial cells of AMI rats.

Features of the Metabolisms of Cardiac Troponin Molecules-Part 1: The Main Stages of Metabolism, Release Stage.

Cardiac troponins (cTns) have long been the most valuable and specific biomarkers for detecting ischemic myocardial cells (MCs) injury, which is one of the key signs of myocardial infarction (MI). Modern methods (highly sensitive and ultra-sensitive immunoassays (hs-cTns)) of detection are an important and indispensable tool for the early diagnosis of MI and the choice of patient management protocols. Timely diagnosis of MI can significantly improve the prognosis of patients. However, in real clinical practice, doctors often face a significant problem when using cTns—the difficulty of differential diagnosis due to frequent and unexplained increases in the concentration of cTns in blood serum. In addition, there is conflicting information that may potentially affect the diagnostic capabilities and value of cTns: the influence of certain biological factors (diurnal rhythm, gender and age) on serum cTns levels; extra-cardiac expression of cTns; the possibilities of non-invasive diagnosis of MI; and other pathological conditions that cause non-ischemic injury to MCs. To solve these problems, it is necessary to concentrate on studying the metabolism of cTns. The review of our current knowledge about cTns metabolism consists of two parts. In this (first) part of the manuscript, the main stages of cTns metabolism are briefly described and the mechanisms of cTns release from MCs are considered in detail.

Post-mortem Gross Detection of Early Myocardial Infarction using Triphenyl Tetrazolium Chloride Test

Background: Ischemic heart disease is the leading cause of mortality in the world. More than 17 millionpeople die each year as a result of myocardial infarctions. Post-mortem histochemical detection of earlymyocardial infarction using 2,3,5-Triphenyl Tetrazolium Chloride (TTC) technique is based on the factthat ischemic myocardial cells lose their membrane integrity and release dehydrogenase enzyme into theblood stream, resulting in decrease or total depletion of these enzymes within the necrotic areas of themyocardium, and so, after immersion in TTC solution, enzyme depleted necrotic myocardium will notretain TTC dye and therefore appears as pale/yellow areas. Materials and Methods: This prospectivepostmortem study was conducted at Medicolegal Directorate in Baghdad city during the period fromMarch 2020 to December 2020. Myocardial specimens were isolated from 75 cases of sudden natural deathand were subjected to 1% TTC solution staining for gross assessment, and subsequently, tissue samples weretaken from suspected areas, processed and stained with H&E for histopathological evaluation and detectionof a possible acute myocardial infarction. Results: The mean age of studied cases was 50.3 ±12.57 years,ranging from 18 to 78 years, median age was 53 years. Males constituted the majority of cases (80%). Caseswere predominantly within 40-59 years age group (58%). The postmortem interval was ranging from 2 to17 hours (mean= 8.6 ±3.6 hours). After immersion in 1% TTC solution, 45 myocardial specimens showedmacroscopic pale/yellow areas which were indicative of acute myocardial infarcts within the ventricularwall, while histopathological examination of specimens revealed features of acute myocardial infarction(AMI) in 62 specimens. TTC stain was found to have a diagnostic sensitivity of 69.4% and specificity of76.9% in postmortem detection of AMI. Conclusion: The TTC technique allows identification of earlymyocardial necrosis. It’s practical, reliable and valid method that promises to be of considerable value. Itcan be used together with histopathology for postmortem detection of visually unapparent acute myocardialinfarcts.

Astragaloside IV: An Effective Drug for the Treatment of Cardiovascular Diseases.

Cardiovascular disease (CVD), the number one cause of death worldwide, has always been the focus of clinical and scientific research. Due to the high number of deaths each year, it is essential to find alternative therapies that are safe and effective with minimal side effects. Traditional Chinese medicine (TCM) has a long history of significant impact on the treatment of CVDs. The mode of action of natural active ingredients of drugs and the development of new drugs are currently hot topics in research on TCM. Astragalus membranaceus is a commonly used Chinese medicinal herb. Previous studies have shown that Astragalus membranaceus has anti-tumor properties and can regulate metabolism, enhance immunity, and strengthen the heart. Astragaloside IV (AS-IV) is the active ingredient of Astragalus membranaceus, which has a prominent role in cardiovascular diseases. AS-IV can protect against ischemic and hypoxic myocardial cell injury, inhibit myocardial hypertrophy and myocardial fibrosis, enhance myocardial contractility, improve diastolic dysfunction, alleviate vascular endothelial dysfunction, and promote angiogenesis. It can also regulate blood glucose and blood lipid levels and reduce the risk of cardiovascular diseases. In this paper, the mechanism of AS-IV intervention in cardiovascular diseases in recent years is reviewed in order to provide a reference for future research and new drug development.

Xuefu Zhuyu Oral Liquid () Prevents Apoptosis of Ischemic Myocardium Cells in Rats by Regulating SIRT1 and Its Pathway-Related Genes.

To observe the changes of ischemic myocardial cells apoptosis in rats following intervention with Xuefu Zhuyu Oral Liquid (, XFZY), as well as changes of protein expression of silent information regulator 1 (SIRT1) and SIRT1 pathway-related genes. H9c2 rat myocardial cells were divided into 6 groups: control group, oxygen glucose deprivation (OGD) group, SIRT1 siRNA group, OGD+SIRT1 siRNA group, OGD+XFZY group, and OGD+SIRT1 siRNA+XFZY group. Quantitative fluorescent polymerase chain reaction (PCR) and Western blot were used to detect the concentration variations of SIRT1 and its pathway-related genes and corresponding protein expression after XFZY intervention and SIRT1 transfection. Compared with the control group, the mRNA and protein expressions of SIRT1 were decreased obviously, while the mRNA and protein levels of P53, FoxO1, FoxO3, FoxO4 and nuclear factor kappa B (NF-ΚB) were increased in the OGD group, SIRT1 siRNA group, and OGD+SIRT1 siRNA group (P<0.01). Compared with the OGD group and OGD+SIRT1 siRNA group, the treatment of XFZY inhibited the decline in SIRT1 mRNA and protein expressions (P<0.01), and down-regulated the mRNA and protein levels of P53, FoxO1, FoxO3, FoxO4 and NF-ΚB, respectively (P<0.05 or P<0.01). XFZY could prevent myocardial cells apoptosis probably by increasing the mRNA and protein expressions of SIRT1 and inhibiting the mRNA and protein expressions of P53, NF- K B, FoxO1, FoxO3 and FoxO4.

Effects of the combination of tanshinone IIA and puerarin on cardiac function and inflammatory response in myocardial ischemia mice

BackgroundVentricular remodeling is a major pathological process of normal heart failure. With the aging of society, poor diet control, social, psychological and other risk factors in our country, the incidence of myocardial infarction and hypertension is reported to increase yearly. Many treatment methods have effectively delayed the occurrence of ventricular remodeling. However, in order to prevent and delay the occurrence and development of ventricular remodeling, the new treatment strategy cannot be ignored. MethodsIn this study, we used male C57BL/6 mice (8 weeks old), weight 23 g–27 g, SPF grade. According to the established methods of the research group, the left anterior descending branch of the coronary artery (LAD) was used to make the model of myocardial ischemia, and which was evaluated by the change of EF value in mice. The experiment included seven groups: sham operation group, model group, metoprolol group, puerarin group, tanshinone IIA group, tanshinone IIA: puerarin =1:1 group, tanshinone IIA: puerarin =1:2 group. The changes of cardiac function in each group were observed by echocardiography and hemodynamics after the drug delivery cycle was 3d, 7d, 14d and 28d. Detection of 3d serum enzyme indexes LDH, CK and CK-MB by automatic biochemical analyzer. The expression of CD11b, F4/80, Ly6C in cardiac tissues were detected by flow cytometry at 3d and 7d. The expression of IL-1β and TNF- α in serum were detected by ELISA. IL-1β, IL-6, IL-10, iNOS and other related genes were detected by RT-PCR method. HE, Masson staining and immunohistochemical staining were used to observe the changes of myocardial histomorphology in mice. We also examined the effects of different drug treatments on the proliferation and function of Raw264.7 cells, H9C2 cells and HUVECs. Western blot examined the effects of different drug treatments on the expression of inflammatory pathway related proteins TLR4 and C/EBP-β. Results1. Echocardiographic results showed that with the prolongation of ischemic time, the ejection fraction of the model group, the shortening rate of the short axis of the left ventricle, the flow rate of the outflow tract were significantly decreased, and the structure of the ventricle was significantly changed. Hemodynamic tests showed that the maximum and maximum rate of decline in the post-ischemia model group were significantly reduced, with increased systolic and diastolic volume, and a decrease in pressure difference. After treatment with drugs, all groups improved, but tanshinone IIA: puerarin = 1:1 group can significantly improve the above indicators after 28d of administration, which can effectively relieve the deterioration of cardiac function caused by acute myocardial infarction. 2. After administration for 3 and 7 days, the inflammatory cell CD11b monocytes and the F4/80 phenotype macrophages in heart tissue were detected by flow cytometry, and it was found that tanshinone IIA: puerarin = 1:1 can inhibit the release of inflammatory cells. The results of RT-PCR showed that the tanshinone IIA: puerarin = 1:1 group significantly improved the expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, and iNOS. In the immunohistochemical analysis of iNOS and Arg-1, the tanshinone IIA and puerarin 1:1 treatment group was able to inhibit the expression of M1 macrophages in the early stage of inflammation and promote the expression of M2 macrophages. 3. The cardiac index increased significantly and the serum TGF-β increased after 28d. The combination of tanshinone IIA and puerarin could significantly reduce these indexes. HE, Masson, Sirius red and immunohistochemical staining were found in the combination of tanshinone IIA and puerarin can significantly reduce the structure of acute ischemic myocardial cell damage and interstitial edema, reduce collagen synthesis, and fibroblasts release, thereby inhibiting myocardial fibrosis and heart remodeling. 4. MTT assay showed a significantly greater proliferation of above two cells types treated with tanshinone IIA: puerarin =1:1 and more nodes and meshes were found in tanshinone IIA: puerarin =1:1 group compared with other groups. 5. The combination of tanshinone IIA and puerarin could regulate inflammation through inhibiting the expression of TLR4 protein, but up-regulating the expression of C/EBP-β protein. ConclusionThe combination of tanshinone IIA and puerarin inhibits the immersion of inflammatory cells. Improving hemodynamics by improving cardiac function, reducing the destruction of cardiac myocytes, reducing collagen synthesis, inhibiting myocardial fibrosis and ventricular remodeling. Through the whole experiment, tanshinone IIA: puerarin = 1:1 is the best.

Exosomes derived from SDF1‐overexpressing mesenchymal stem cells inhibit ischemic myocardial cell apoptosis and promote cardiac endothelial microvascular regeneration in mice with myocardial infarction

Exosomes extracted from mesenchymal stem cells (MSCs) was reported to reduce myocardial ischemia/reperfusion damage. Besides, stromal-derived factor 1 (SDF1a) functions as cardiac repair after myocardial infarction (MI). Therefore, the present study aims to identify whether exosomes (Exo) released from SDF1-overexpressing MSCs display a beneficial effect on ischemic myocardial infarction. Initially, a gain-of-function study was performed to investigate the function of SDF1 in ischemic myocardial cells and cardiac endothelial cells. Coculture experiments were performed to measure potential exosomic transfer of SDF1 from MSCs to ischemic myocardial cells and cardiac endothelial cells. During the coculture experiments, exosome secretion was disrupted by neutral sphingomyelinase inhibitor GW4869 and upregulated exosomal SDF1 using SDF1 plasmid. Effects of Exo-SDF1 on cardiac function in MI mice were investigated in vivo. MSCs suppressed myocardial cell apoptosis and promoted microvascular regeneration of endothelial cells through secretion of exosomes. The addition of GW4869 led to increased apoptotic capacity of myocardial cells, decreased microvascular formation ability of endothelial cells, enhanced autophagy ability, and elevated Beclin-1 level as well as ratio of LC3II/LC3I. Overexpression of SDF1 and Exo-SDF1 inhibited apoptosis and autophagy of myocardial cells, but promoted tube formation of endothelial cells. The interference of PI3K signaling pathway promoted apoptosis and autophagy of myocardial cells, but inhibited tube formation of endothelial cells. SDF1 activated the PI3K signaling pathway. Exo-SDF1 protected cardiac function of MI mice and inhibited myocardial tissue damage. This study provided evidence that SDF1 overexpression in MSCs-derived exosomes inhibited autophagy of ischemic myocardial cells and promoted microvascular production of endothelial cells.

Stimulation of autologous progenitorial and committed cells in ischemically damaged myocardium

Summary. Local progenitor cells are present in all tissues, including the myocardium. Stimulation of these cells can significantly improve the efficiency of the regeneration process. Biodegradation products of Alloplant biomaterials (ABM) are an inhibitor of fibrosis and serve as an inducer of stem cells in various body tissues including smooth and skeletal muscles, as well as connective tissue.Aim. To determine the cell regenerative potential of progenitorial c-kit and committed GATA-4 cells in ischemic myocardium on the background of ABM application in chronic experiment.Materials and methods. 100 Wistar rats weighing 180-200g were used, that underwent ligation of coronary artery. In the experimental group (n = 50), 600 μl of a physiological solution containing 12 mg of ABM was administered simultaneously with coronary occlusion. General histological studies (hematoxylin and eosin staining, and staining as per Mallory), immunohistochemical (c-kit, GATA-4) and morphometric studies (determination of the index of the scar's area, counting of stained cells) were carried out. The statistical processing of the results was carried out using the rank methods - single factor dispersion analysis as per Kruskalou-Wallace and the comparison of uncorrelated data by the Mann-Whitney method.Results. When using ABM during coronary occlusion, myocardium was characterized by a statistically significant (p &lt;0.0001) prevalence of c-kit+ / GATA-4+ cells numbers throughout the follow-up period (3-45 days). These cells were localized mainly in the granulation tissue of the peri-cicatricial area, bordering on the preserved cardiac muscle tissue. In the peri-cicatricial area, cardiac cells had a different degree of maturity - from poorly differentiated forms (without clear ultrastructural signs of cardiomyogenic orientation) to young cardiomyocytes. In the presence of ABM, coronary occlusion was characterized by statistically significantly smaller values of the index of the scar's area (p &lt;0,05 ÷ &lt;0,0001).Conclusion. ABM degradation products are chemoattractants for c-kit cells and promote their differentiation into cardiomyogenic GATA-4 cells, which contributes to more successful myocardium repair.

Possible role of circulating endothelial cells in patients after acute myocardial infarction

Acute myocardial infarction (AMI) occurs as a result of insufficient myocardial perfusion leading to cell necrosis. This is most commonly due to the obstruction of the coronary artery by ruptured atherosclerotic plaque and thrombosis. Damaged ischemic and necrotic myocardial cells release pro-inflammatory substances in tissue and plasma, leading to a systemic inflammatory response. Profound systemic inflammatory response during ischemia/reperfusion injury causes disruption of endothelial glycocalyx and detachment of endothelial cells that express von Willebrant factor (vWF). We hypothesize that circulating vWF+ endothelial cells could act as antigen presenting cells which interact with T and NK cells directly, by cell to cell contact and indirectly by cytokine and chemokine secretion, leading to the immune response towards inflammation. Analyzing the frequency, phenotype and pro-inflammatory substances produced in circulating vWF positive (+) cells in patients with AMI could be beneficial to determine the severity of the pro-inflammatory response, according to the level of endothelial dysfunction in the early period of AMI. To evaluate these hypotheses, we suggest to determine frequency, phenotype, and ability of cytokine/chemokine production in circulating vWF+ endothelial cells by simultaneous surface and intracellular cell staining, and flow cytometry analysis. Secretion of pro-inflammatory cytokines and chemokines, pro-atherogenic substances and the components of glycocalyx might be measured in supernatants of magnetically separated or sorted vWF+ endothelial cells, as well as in the serum of a patient with acute AMI by enzyme linked–immunoassay tests. The interaction of increasing concentrations of isolated circulating vWF+ endothelial cells and cognate T and NK cells might be investigated by lymphocyte proliferation rate, cytotoxic mediators’ expression, and cytokine production. If our hypothesis is correct, characterization of circulating vWF+ endothelial cells could grant us greater insight into their role in pathophysiology of AMI and the degree of myocardial damage.

Rac1 promotes the survival of H9c2 cells during serum deficiency targeting JNK/c-JUN/Cyclin-D1 and AKT2/MCL1 pathways.

Rac1, known as a “molecular switch”, plays a crucial role in plenty of cellular processes. Rac1 aggravates the damage of myocardial cells in the process of myocardial ischemia-reperfusion during myocardial infarction through activating the NADPH oxidase and bringing about the reactive oxygen species(ROS) generation. Myocardial ischemia and hypoxia are the basic pathogenesis of myocardial infarction and the underlying mechanisms are intricate and varied. Moreover, the regulatory effect of Rac1 on myocardial cells in the condition of serum starvation and the potential mechanisms are still incompletely undefined. Therefore, heart-derived H9c2 cells cultured in 0% serum were used to mimic ischemic myocardial cells and to clarify the role of Rac1 in H9c2 cells and the underlying mechanisms during serum deficiency. After Rac1 was knocked down using specific siRNA, cell apoptosis was assessed by flow cytometry assay and cell proliferation was detected by CCK-8 assay and EdU assay. In addition, the expression and activation of protein in related signaling pathway were detected by Western blot and siRNAs was used to testify the signaling pathways. Our results indicated that Rac1 inhibited apoptosis, promoted proliferation and cell cycle progression of H9c2 cells during serum deficiency. We concluded that Rac1 inhibited apoptosis in an AKT2/MCL1 dependent way and promoted cell proliferation through JNK/c-JUN/Cyclin-D1.

Diagnosis of Early Myocardial Infarction by Histochemical Staining of Heart on Autopsy

INTRODUCTION : The incidence of Sudden Unexpected Death is increasing globally. Cardio vascular disease is the most common cause for sudden death. Eighty percent of sudden cardiac death is due to coronary arterial disease36. In 25% of cases death occurs abruptly and unexpectedly within first hour of onset of clinical symptom. Most often, the identification of early change of Myocardial infarction becomes difficult during postmortem examination, as gross change of infarct will not be apparent for 24 to 48 hrs following myocardial ischemic damage Histochemical staining techniques using azo dyes are based on the fact that ischemic myocardial cells lose their membrane integrity and release their enzyme contents in to the blood, resulting in a marked decrease or total depletion of these enzymes in the ischemic areas of the myocardium. Enzyme depleted infarct myocardium remain unstained, which is the principle of this study. AIMS AND OBJECTIVES : 1. To diagnose early Myocardial Infarction by Histochemical staining of Myocardium using Triphenyl Tetrazolium Chloride (TTC) while performing autopsy, in the absence of appreciable macroscopic changes in the myocardial tissue. 2. To confirm those identified areas of early myocardial infarction by Histopathological Examination. 3. To determine the diagnostic validity of the Histochemical Staining (TTC) of heart in gross detection of early Myocardial Infarction. MATERIALS AND METHODS: Slices from 18 hearts of suggested or suspected sudden cardiac death were incubated in 1% Triphenyl Tetrazolium Chloride Solution for 20 min. after adjusting the pH of the solution using phosphate buffer. The heart slices were examined for unstained area. Normal myocardium was stained whereas infarct areas remain unstained. Both stained and unstained areas were subjected to Histopathological examination for early Myocardial Infarction. OBSERVATION AND RESULTS: Of 18 hearts stained with TTC solution, 11 hearts showed positive TTC result, 5 hearts showed negative result, 1 heart showed false positive result and 1 heart showed false negative result. CONCLUSION : Histochemical staining using 1% TTC solution is a reliable method in detection of early myocardial infarction. Though this method has diagnostic validity of 88.88%, it could not identify death due to arrhythmias.

Beneficial effects of Schisandrin B on the cardiac structure and function in a mice model of myocardial infarction

Objective: To investigate whether Schisandrin B (Sch B) could improve cardiac structure and function in myocardial infarction (MI) mice and related mechanisms. Methods: Male C57BL/6J mice were randomized into sham (n=8), MI+ Sch B (n=24, 80 mg·kg(-1)·d(-1) per gavage) or MI+ vehicle (n=24, equal volume). After treatment for 3 weeks, cardiac function was detected by echocardiography measurement.Infarction size was measured by Evans blue and TTC staining.HE and Masson trichrome staining were used to observe the myocardial inflammation, structure and fibrosis.TNF-α, TGF-β, IL-1β were detected by ELISA. The apoptosis index of ischemic myocardial cells was detected by immunofluorescence. NF-κB, Bcl-2, Bax, Akt phosphorylated Akt(p-Akt), eNOS, phosphorylated eNOS (p-eNOS) were detected by Western blot. Results: Three weeks after operation, survival rate (83.33% vs. 54.17%, P<0.05), LVEF((51.77±8.50)% vs.(40.23±8.30)%, P<0.05), LVFS((26.44±5.15)% vs. (19.53±4.56)%, P<0.05)were significantly higher; LVEDD ((4.13±0.40) mm vs.(4.44±0.46)mm, P<0.05), LVESD((3.07±0.39) mm vs. (3.46±0.52)mm, P<0.05), the heart weight/body weight ratio((0.59±0.06)% vs. (0.68±0.10)%, P<0.05)was significantly lower and infarct size ((23.4±2.36)% vs. (39.4±2.06)%, P<0.05) was significantly reduced in MI+ Sch B group than those in MI+ vehicle group. In MI+ vehicle group, HE staining showed a large number of inflammatory cells in the peri-infarctl region, and the permutation structure was very disorganized, while above changes were significantly reduced in the MI+ Sch B group. Masson staining results showed that the degree of myocardial fibrosis in MI+ Sch B group was significantly less than that of MI+ vehicle group.Moreover, Sch B could down-regulate some inflammatory cytokines, like NF-κB、TGF-β、TNF-α and IL-1β, activate Akt-eNOS pathway, upgrade Bcl-2 and downgrade Bax and reduce cell apoptosis as compared with MI+ vehicle group (all P<0.05). Conclusions: Our results demonstrate that Sch B can reduce inflammation, inhibit apoptosis, and attenuate cardiac remodeling and improve cardiac function in this mice MI model.Sch B might serve as a potential novel therapeutic agent for ischemic heart disease.

Abstract 24: Graphene Oxide Containing Thermosensitive Hydrogel Improves the Survival of Transplanted Stem Cells in Ischemic Myocardium

Bone marrow derived mesenchymal stem cell (MSC) therapy has the potential to preserve cardiac function and prevent heart failure following a myocardial infarction (MI). However, in our recent studies, long term survival of transplanted stem cells was not detected, which was associated with deterioration of cardiac function. Therefore, strategies to enhance survival of transplanted cells would preserve the benefits of MSCs therapy for heart repair. Recently, graphene oxide (GO) nanosheets are being widely investigated for their ability to promote adherence and homing of transplanted MSCs to the extra cellular matrix in the heart. However, the presence of reactive oxygen functional groups (-OH, -COOH) in the structure of GO increases oxidative stress and activate apoptotic pathways in MSCs, which impairs the benefits of GO based biomaterials. Therefore, to nullify these side effects of GO, we synthesized a novel thermosensitive hydrogel by conjugating GO with chitosan (CS). Our Fourier transformed infrared spectrum (FTIR) analysis revealed an interaction between the oxygen functional groups of GO and amino (-NH2) groups of chitosan. Our data also demonstrate that CS masked the reactive functional groups of GO and prevented GO mediated ROS induction. This novel chitosan-graphene oxide (CS-GO) composite exhibited optimal porosity for cell conjugation and retention. Furthermore, this novel biomaterial was cyto-compatible as it exerted negligible toxicity to encapsulated MSCs. Upon exposure to hypoxic/ischemic environment, we observed a significant increase in apoptosis in MSCs as evidenced by an increase in the Bax/Bcl-xl ratio and caspase 3 activation. However, conjugation of MSCs to CS-GO polymer prevented hypoxia induced apoptosis. Therefore, coupling of GO with chitosan prevented GO mediated oxidative stress and downregulated hypoxia induced apoptosis of MSCs. Therefore, the outcome of this study will provide a novel delivery system for MSCs to the heart and improve their long term survival in the infarcted heart.

Activation of cannabinoid receptor type II by AM1241 protects adipose-derived mesenchymal stem cells from oxidative damage and enhances their therapeutic efficacy in myocardial infarction mice via Stat3 activation.

The poor survival of cells in ischemic sites diminishes the therapeutic efficacy of stem cell therapy. Previously we and others have reported that Cannabinoid receptor type II (CB2) is protective during heart ischemic injury for its anti-oxidative activity. However, whether CB2 activation could improve the survival and therapeutic efficacy of stem cells in ischemic myocardium and the underlying mechanisms remain elusive. Here, we showed evidence that CB2 agonist AM1241 treatment could improve the functional survival of adipose-derived mesenchymal stem cells (AD-MSCs) in vitro as well as in vivo. Moreover, AD-MSCs adjuvant with AM1241 improved cardiac function, and inhibited cardiac oxidative stress, apoptosis and fibrosis. To unveil possible mechanisms, AD-MSCs were exposed to hydrogen peroxide/serum deprivation to simulate the ischemic environment in myocardium. Results delineated that AM1241 blocked the apoptosis, oxidative damage and promoted the paracrine effects of AD-MSCs. Mechanistically, AM1241 activated signal transducers and activators of transcription 3 (Stat3) through the phosphorylation of Akt and ERK1/2. Moreover, the administration of AM630, LY294002, U0126 and AG490 (inhibitors for CB2, Akt, ERK1/2 and Stat3, respectively) could abolish the beneficial actions of AM1241. Our result support the promise of CB2 activation as an effective strategy to optimize stem cell-based therapy possibly through Stat3 activation.

Effect of total peony glucoside pretreatment on NF-κB and ICAM-1 expression in myocardial tissue of rat with myocardial ischemia-reperfusion injury

Early recovery of myocardial perfusion is beneficial for myocardial ischemia. However, ischemia-reperfusion (I/R) may exacerbate myocardial injury. Research shows that total peony glucoside (TPG) can inhibit ischemic myocardial cell apoptosis. However, whether it can ameliorate I/R injury remains poorly understood. This study explored the effect of TPG pretreatment on I/R, through nuclear factor-kappa B (NF-κB) and intercellular adhesion molecule-1 (ICAM-1) expressions in I/R-affected myocardium. Healthy 7-week-old male Sprague Dawley rats were randomly categorized into sham operation (A), modeling (B), and 100, 200, and 400 mg/kg TPG pretreatment groups (C, D, and E, respectively), with 20 rats in each group. I/R rat models were designed by ligating left anterior descending coronary artery for 30 min to induce ischemia and for 120 min to induce reperfusion. Serum interleukin 6 (IL-6) and interleukin 8 (IL-8) levels were measured using enzyme linked immunosorbent assay. NF-κB and ICAM-1 mRNA and protein expressions were detected through RT-PCR and western blot analysis, respectively. Compared to group A, serum IL-6 and IL-8 levels of group B elevated significantly (P < 0.05), whereas NF-κB and ICAM-1 mRNA and protein expressions increased in the myocardium (P < 0.05). Serum IL-6 and IL-8 levels, and NF-κB and ICAM-1 mRNA and protein expressions, in myocardium of TPG groups reduced in a dose-dependent manner. Therefore, TPG pretreatment could alleviate myocardium reperfusion injury in I/R rat models by reducing NF-κB and ICAM-1 mRNA and protein expressions and cytokine secretions. This mechanism could be associated with the inhibition of NF-κB activation and downregulation of ICAM-1 expression.

Nanoparticles-Assisted Stem Cell Therapy for Ischemic Heart Disease.

Stem cell therapy has attracted increasing attention as a promising treatment strategy for cardiac repair in ischemic heart disease. Nanoparticles (NPs), with their superior physical and chemical properties, have been widely utilized to assist stem cell therapy. With the help of NPs, stem cells can be genetically engineered for enhanced paracrine profile. To further understand the fate and behaviors of stem cells in ischemic myocardium, imaging NPs can label stem cells and be tracked in vivo under multiple modalities. Besides that, NPs can also be used to enhance stem cell retention in myocardium. These facts have raised efforts on the development of more intelligent and multifunctional NPs for cellular application. Herein, an overview of the applications of NPs-assisted stem cell therapy is given. Key issues and future prospects are also critically addressed.

GW26-e2442 Impact of XueFuZhuYu oral liquid over FOXO family expression during ischemic myocardial cells apoptosis

GW26-e2442 Impact of XueFuZhuYu oral liquid over FOXO family expression during ischemic myocardial cells apoptosis