Proliferation Of Myocardial Cells Research Articles

Expression of microRNA-590 in patients with chronic coronary heart disease, atrial fibrillation, and their combination

Background. The role of microRNAs in the processes of remodeling, proliferation, and fibrogenesis of myocardial cells is not clear enough.
 Aim. Analysis of the microRNA-590 expression level in patients with chronic coronary heart disease, atrial fibrillation, as well as their combination to assess the potential prognostic significance of the expression level.
 Material and methods. The study included 94 patients divided into three clinical groups: the first with non-valvular atrial fibrillation without coronary heart disease (39 people); the second with non-valvular atrial fibrillation and coronary heart disease (22 patients); the third with ischemic heart disease without atrial fibrillation (23 patients). The comparison group consisted of 10 people without atrial fibrillation and coronary heart disease. Venous blood was taken from all subjects, from the plasma of which microRNA was isolated. The relative level of microRNA expression was estimated based on real-time polymerase chain reaction data obtained during the reaction on a cycler using commercial TaqMan probes and primers. The statistical significance of differences between groups was determined using one-way analysis of variance, followed by post-hoc analysis using Tukey's contrasts, differences were considered statistically significant at p 0.05. The ShapiroWilk test was used to assess the normality of the distribution of residuals.
 Results. A statistically significant decrease in the expression level of microRNA-590 was registered in the third (p=0.0104) and in the second (p=0.0046) groups compared with the control group, as well as in patients with atrial fibrillation and left atrial dilatation (p=0.0313), with recurrent arrhythmias after radiofrequency ablation (p=0.0083) and with permanent atrial fibrillation (p=0.0242).
 Conclusion. Ischemic heart disease, including when combined with atrial fibrillation, and aggravating factors, such as left atrial dilatation, permanent atrial fibrillation, recurrence of arrhythmia after radiofrequency ablation, lead to a decrease in the expression level of microRNA-590.

Synergetic EGCG and coenzyme Q10 DSPC liposome nanoparticles protect against myocardial infarction.

At the site of myocardial infarction (MI), various phenomena such as oxidative stress and myocardial apoptosis can be observed. Both epigallocatechin gallate (EGCG) and coenzyme Q10 (CoQ10) exhibit antioxidant and anti-inflammatory effects. Macrophages have demonstrated a higher internalization rate of cationic liposomes, thereby increasing their bioavailability. This study utilized EGCG in synergy with CoQ10 as an antioxidant agent and distearyl phosphatidylcholine (DSPC) as the carrier, to create liposome nanoparticles known as CE-LNPs. The CE-LNPs exhibited favorable biocompatibility and were effectively engulfed by macrophages in vitro. In addition, the CE-LNPs effectively eradicated reactive oxygen species (ROS) in hypoxic cardiomyocytes, mitigated myocardial cell apoptosis, and sustained the functionality and proliferation of myocardial cells. The anti-apoptotic effect of the CE-LNPs was further validated through TUNEL and Annexin V FITC/PI experiments. The therapeutic efficacy of CE-LNPs was evaluated in a murine model of MI. CE-LNPs demonstrated a significant reduction in scar area in vivo, facilitating cardiac repair and improving cardiac function. These findings provide evidence that EGCG synergistically combined with CoQ10 in DSPC liposome nanoparticles offers protection against MI.

Hypoxia-elicited cardiac microvascular endothelial cell-derived exosomal miR-210–3p alleviate hypoxia/reoxygenation-induced myocardial cell injury through inhibiting transferrin receptor 1-mediated ferroptosis

ObjectiveFerroptosis is a novel mode of non-apoptotic cell death induced by build-up of toxic lipid peroxides (lipid-ROS) in an iron dependent manner, which is a key event in ischemia/reperfusion (I/R)-induced cardiomyocytes damages. Studies indicated that ischemic preconditioning with cardiac microvascular endothelial cells (CMECs) protected against I/R-induced cardiomyocytes damages. However, the role of hypoxia-conditioned CMECs-derived Exo (H-exo) in I/R cardiomyocytes damages remains largely unclear. Therefore, the objective of this study was to explore the role and underlying mechanisms of H-exo in hypoxia/reoxygenation(H/R)-induced H9C2 cells damages. MethodsThe rat CMECs were subjected to hypoxia or normoxia culture and Exo was subsequently collected and identified. H-exo or normoxia-conditioned CMECs-derived Exo (N-exo) were administered to H9C2 cells with H/R. To evaluate the therapeutic effect of H-exo and H-exo on H/R-induced H9C2 cells damages, cell proliferation was detected by CCK-8 assay and Edu staining, and ferroptosis process were evaluated by iron ion concentration, lipid reactive oxygen species (ROS) level, malondialdehyde (MDA) level, glutathione peroxidase (GSH-Px) level, and the protein expression of ferroptosis markers. Mechanically, we utilized the RT-qPCR to identify the expression of candidate miR-210–3p in N-exo and H-exo. Bioinformatics combined with dual luciferase reporter assay disclosed the downstream molecular mechanism of miR-210–3p. ResultsThe results indicated that both H-exo and N-exo significantly facilitated cell proliferation, increased GSH-Px levels and ferroptosis marker (GPX4) protein levels, and reduced iron ion concentration, lipid ROS level, MDA levels and ferroptosis markers (ACSL4 and PTGS2) protein levels in H/R-treated H9C2 cells. More importantly, the therapeutic effect of H-exo was significantly better than that of N-exo. Mechanistically, the results of RT-qPCR revealed significant enrichment of miR-210–3p in H-exo compared with N-exo. The miR-210–3p delivered by H-exo inhibited TFR expression by directly interacting with TFR mRNA, resulting in the promotion of cell proliferation and the attenuation of cell ferroptosis in H/R-treated H9C2 cells. ConclusionAll these data demonstrated that H-exo derived miR-210–3p facilitated the proliferation of myocardial cells in H/R-treated H9C2 cells by suppressing TFR-mediated ferroptosis, which provided new methods to treat H/R-induced myocardial injury.

Diflubenzuron Induces Cardiotoxicity in Zebrafish Embryos.

Diflubenzuron is an insecticide that serves as a chitin inhibitor to restrict the growth of many harmful larvae, including mosquito larvae, cotton bollworm and flies. The residue of diflubenzuron is often detected in aquaculture, but its potential toxicity to aquatic organisms is still obscure. In this study, zebrafish embryos (from 6 h to 96 h post-fertilization, hpf) were exposed to different concentrations of diflubenzuron (0, 0.5, 1.5, 2.5, 3.5 and 4.5 mg/L), and the morphologic changes, mortality rate, hatchability rate and average heart rate were calculated. Diflubenzuron exposure increased the distance between the venous sinus and bulbar artery (SV-BA), inhibited proliferation of myocardial cells and damaged vascular development. In addition, diflubenzuron exposure also induced contents of reactive oxygen species (ROS) and malondialdehyde (MDA) and inhibited the activity of antioxidants, including SOD (superoxide dismutase) and CAT (catalase). Moreover, acridine orange (AO) staining showed that diflubenzuron exposure increased the apoptotic cells in the heart. Q-PCR also indicated that diflubenzuron exposure promoted the expression of apoptosis-related genes (bax, bcl2, p53, caspase3 and caspase9). However, the expression of some heart-related genes were inhibited. The oxidative stress-induced apoptosis damaged the cardiac development of zebrafish embryos. Therefore, diflubenzuron exposure induced severe cardiotoxicity in zebrafish embryos. The results contribute to a more comprehensive understanding of the safety use of diflubenzuron.

CPE Regulates Proliferation and Apoptosis of Primary Myocardial Cells Mediated by Ischemia and Hypoxia Injury.

Objective To observe the effect of carboxypeptidase E (CPE) on the ischemia and hypoxia (I/H) injury of primary cardiomyocytes. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) technology was used to detect the expression of CPE in sham and myocardial infarction (MI) rat heart tissue, and the plasmid was transferred into primary cardiomyocytes by transfection technology. The apoptosis rate of cardiomyocytes was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, Annexin V-PI staining, and Cell Counting Kit-8 (CCK-8) assay. In addition, Caspase kit and qRT-PCR technology were used to detect the expression of apoptosis-related factors. The cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) staining, flow cytometry, and qRT-PCR technology. In addition, Western blotting (WB) and qRT-PCR techniques were used to detect the Wnt/β-catenin pathway. Results First, we found that the expression of CPE in the marginal zone of MI was obviously reduced. Overexpression of CPE in primary cardiomyocytes can effectively inhibit ischemia/hypoxia (I/H)-induced apoptosis and decreased cell activity. In addition, CPE can promote cell proliferation and relieve the inhibitory effect of I/H on cardiomyocytes. At the same time, CPE can promote the expression of β-catenin and c-myc. Conclusion Overexpression of CPE in primary cardiomyocytes can effectively alleviate the decreased cell activity, increased apoptosis, and decreased proliferation caused by I/H and regulated by Wnt/β-catenin pathway.

Serum Extracellular Vesicles Attenuate Cardiomyocyte Injury Induced by Hypoxic/Reoxygenation by Regulating miR-1229-5p.

Ischemic heart disease and the resulting heart failure remain the leading causes of death and disability worldwide. This study aimed to investigate the role of miR-1229-5p in serum extracellular vesicles (EVs) mediated myocardial protection by constructing a hypoxia/reoxygenation model (HR) in H9c2 cells. Cardiomyocytes were cultured and divided into different treatment groups: control group, HR group, serum-EVs group, and serum-EVs + miR-1229-5p inhibitor group. The expression levels of miR-1229-5p were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The changes in cell proliferation and apoptosis were detected by MTT assay and flow cytometry. The myocardial injury-related indicators, cardiac troponin I (cTnI), creatinine kinase MB (CK-MB), and lactate dehydrogenase (LDH), were measured by enzyme-linked immunosorbent assay (ELISA). Finally, the luciferase reporter assay was used to verify the miR-1229-5p target. The proliferation of myocardial cells in the HR group was reduced, the number of apoptotic cells was increased, and myocardial injury indicators concentration was decreased. Transfection of miR-1229-5p inhibitor under serum-EVs treatment reduced the protective effect of serum-EVs on myocardial cell injury, decreased cell proliferation, increased the number of apoptotic cells, and increased myocardial injury indicator concentration. Additionally, FOXO4 may be the target of miR-1229-5p. Our data suggest that serum-EVs alleviate HR-induced cardiomyocyte injury by regulating miR-1229-5p/FOXO4.

In vitro and in vivo studies on the biocompatibility of a self-powered pacemaker with a flexible buckling piezoelectric vibration energy harvester for rats.

BackgroundScavenging energy from biomechanical motions in vivo by energy converting devices, i.e., implantable harvesters, to obtain sustainable electrical energy is the ideal way to power implantable medical devices which require long term and continuous power supply. A novel self-powered cardiac pacemaker is designed to achieve self-powered pacing. The kinetic energy of the heart was collected by an implanted piezoelectric energy collector and supplied to the cardiac pacemaker, and then the cardiac tissue was stimulated by the pacing electrode pierced from the outside of the heart to realize effective pacing effect and self-powered pacing. In this study, we evaluated the stability and biocompatibility of our previously described flexible buckling piezoelectric vibration energy harvester in vitro and in vivo. The biocompatibility, in vivo stability, and safety of the self-powered pacemaker with a flexible flexion piezoelectric vibratory energy harvesting device prepared were analyzed by performing cell and in vivo animal experiments.MethodsThe MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to detect the cell proliferation of H9C2 cells and HUVECs at 24, 48, and 72 hours. Computed tomography (CT) and cardiac ultrasound were used to evaluate the position and heart rate of pacemakers 12 weeks after implantation, and the changes of plasma biochemical indexes were detected by a biochemical detector.ResultsAt 12 weeks after implantation, CT results showed that there were no changes in the position of the self-powered pacemaker. The device implanted into the thoracic cavity of rats demonstrated certain effects on cardiac function, while it did not have a significant effect on their blood biochemical indexes.Conclusionsthe flexible buckling piezoelectric vibratory energy collector did not produce adverse effects on the myocardial tissue or on the normal proliferation of myocardial cells.

Protective effect of MAPK signaling pathway mediated by ITGB3 gene silencing on myocardial ischemia-reperfusion injury in mice and its mechanism.

To explore the effect of integrin β3 (ITGB3) gene silencing mediated mitogen-activated protein kinase (MAPK) signaling pathway on myocardial ischemia-reperfusion injury (MIRI) in mice. MIRI mice model was established, and myocardial tissues of MIRI mice and sham operation group mice were extracted. Hematoxylin-Eosin (HE) staining was used to observe the pathological changes of myocardial tissue; terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to detect the apoptosis of myocardial cells; ELISA method was used to detect the levels of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α in the two groups. The infarct size was measured by TTC staining. Myocardial cells of MIRI model mice were isolated and cultured, and then grouped and transfected. The cells were transfected with the grouping of MIRI group, negative control (NC) group, MAPK signal pathway agonist Anisomycin group, MAPK signal pathway inhibitor SB203580 group, ITGB3-siRNA group, SB203580 + ITGB3-siRNA group. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to detect the mRNA and protein expressions of ITGB3, p38MAPK/p-p38MAPK, GSK-3β/p-GSK-3β, Cx43/p-Cx43, pro-apoptotic factor Bax and anti-apoptotic factor Bcl-2. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was used to detect cell proliferation and flow cytometry to detect cell apoptosis. The expression of ITGB3 in myocardial tissue of MIRI mice was significantly higher than that of sham operated mice (p<0.05). Compared with the sham operation group, the apoptosis rate of myocardial cells in MIRI group was significantly increased, the expression levels of IL-1, IL-6 and TNF-α were significantly increased, and the myocardial infarction area was significantly increased (all p<0.05). Compared with MIRI and NC groups, ITGB3 mRNA and protein expression levels in ITGB3-siRNA group and SB203580 + ITGB3-siRNA group were significantly decreased (all p<0.05), but no significant change was found in Anisomycin group and SB203580 group (p>0.05). Furthermore, ITGB3-siRNA group and Anisomycin group had markedly decreased mRNA and protein expressions of ITGB3 and Bax, increased mRNA and protein expressions of p38MAPK/p-p38MAPK, GSK-3β/p-GSK-3β, Cx43/p-Cx43 and Bcl-2, as well as increased cell proliferation and decreased cell apoptosis (all p<0.05); SB203580 group indicated an opposite result with Anisomycin group; while SB203580 + ITGB3-siRNA revealed none significant statistical difference. In addition, compared with ITGB3-siRNA group, SB203580 + ITGB3-siRNA group showed significantly upregulated mRNA and protein expressions of Bax, downregulated mRNA and protein expressions of p38MAPK/p-p38MAPK, GSK-3β/p-GSK-3β, Cx43/p-Cx43 and Bcl-2, as well as decreased cell proliferation and increased cell apoptosis (all p<0.05). Silencing ITGB3 gene expression can promote the activation of MAPK signaling pathway, elevate the phosphorylation of GSK-3β and Cx43 in the downstream, promote the proliferation of mouse myocardial cells, inhibit myocardial cell apoptosis and inflammatory reaction, and thus have protective effect on MIRI in mice.

LncRNA-P21 suppresses apoptosis of myocardial cells in rats with acute myocardial infarction via regulating Wnt/β-catenin signaling pathway.

The aim of this study was to investigate the expression of long non-coding ribonucleic acid (lncRNA)-p21 in rats with acute myocardial infarction (AMI) and its influences on the viability and apoptosis of myocardial cells. Sprague-Dawley rats were utilized to establish the AMI model. Myocardial tissues were extracted, and myocardial cells were isolated. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of lncRNA-p21. Subsequently, myocardial cells of AMI rats were cultured and transfected with lncRNA-p21 or small interfering (si)-lncRNA-p21. 48 h later, cell proliferation was determined using Cell Counting Kit-8 (CCK-8). Caspase-3 kit was applied to examine the changes in Caspase-3 after myocardial cell transfection. Moreover, Western blotting assay was performed to measure the protein expressions of apoptosis-associated indexes [B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax)] and key members of the Wnt signaling pathway (Wnt-5a and β-catenin). The expression of lncRNA-p21 decreased significantly in myocardial tissues (p<0.01). However, the mRNA expression of inflammatory factors was significantly upregulated in AMI group compared with those in Sham group (p<0.01). Compared with Vehicle group, LncRNA-p21 group exhibited remarkably enhanced myocardial cell viability (p<0.05). However, Si-lncRNA-p21 group weakened myocardial cell viability (p<0.05). The expression of Caspase-3 in myocardial cells was reduced in LncRNA-p21 group (p<0.05), but remarkably rose in Si-lncRNA-p21 group (p<0.05) in comparison with Vehicle group. In addition, the protein expressions of Wnt-5a and β-catenin in myocardial cells were significantly higher in LncRNA-p21 group (p<0.05), whereas were lower in Si-lncRNA-p21 group (p<0.05) than those in Vehicle group. LncRNA-p21 was lowly expressed in myocardial tissues of AMI rats. Furthermore, it affected the proliferation, apoptosis and inflammation level of myocardial cells in AMI rats by activating the Wnt/β-catenin signaling pathway.

MiR-30b-5p promotes myocardial cell apoptosis in rats with myocardial infarction through regulating Wnt/β-catenin signaling pathway.

The aim of this study was to screen differentially expressed microribonucleic acids (miRNAs) in the heart tissues of rats with myocardial infarction (MI), to explore the relationship between miR-30b-p and MI, and to further research the action mechanism of miR-30b-p. Rat MI models were established. Then 3 rats in experimental (MI) group and 3 rats in sham operation (Sham) group were selected, RNAs were extracted from heart tissues, and differentially expressed miRNAs in the two groups were screened using transcriptome sequencing techniques. TargetScan (http://www.targetscan.org/vert_80/) was applied to predict miR-30b-5p target genes, which then were collected and subjected to enrichment analysis conducted using Metascape (Metascape, Berlin, Germany). Myocardial cells were extracted from MI rats and transfected with miR-30b-5p inhibitor. Then the changes in cell proliferation and apoptosis were detected by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay and flow cytometry, respectively. The expressions of β-actin, β-catenin, Caspase-3 and cleaved Caspase-3 were detected in myocardial cells transfected with miR-30b-5p via Western blotting (WB). A total of 85 differentially expressed miRNAs were detected in the heart of MI rats, and miR-30b-5p expression was down-regulated (P<0.01). In addition, target genes regulated by miR-30b-5p participated in the vascular development, miRNA metabolic process and Wnt signaling pathway. In comparison with miR-negative control (NC) transfection, miR-30b-5p inhibitor transfection promoted the proliferation of myocardial cells but inhibited their apoptosis (P<0.05). Compared with those in miR-NC transfection group, the expressions of β-catenin and cleaved Caspase-3 proteins were down-regulated in miR-30b-5p inhibitor transfection group (P<0.01). MiR-30b-5p is involved in the proliferation and apoptosis of myocardial cells via regulating the Wnt/β-catenin signaling pathway, and it may become a potential target for the diagnosis of MI in clinic.

Effects of Promethazine on Proliferation and Apoptosis of Myocardial Cells in Rats with Myocardial Ischemia-Reperfusion Injury Through PI3K/Akt Signaling Pathway

Objective: To assess promethazine's effect on myocardial cells in rats with myocardial ischemiareperfusion injury (MIRI). Methods: The rat MIRI model was established and treated as the ischemia group. MIRI rats were treated with promethazine and included as the drug group. Rats only undergoing thoracotomy were enrolled as the control group. The physiological function of heart was assessed using the ultrasound cardiotachograph, and the apoptosis and proliferation of myocardial cells were detected using TUNEL assay and Ki67 staining, respectively. Moreover, the expressions of Caspase-3, Bcl-2, PI3K, GSK-3, PDK-1 and PKB were determined via Western blotting and qPCR. Results: There were significant differences in cardiac function indexes [left ventricular enddiastolic diameter (LVEDd), left ventricular end systolic diameter (LVESd), ejection fraction (EF) and fractional shortening (FS)] among the three groups (p= 0 002, 0.004, 0.025 and 0.012), and ischemia group had the highest LVEDd [(8.73± 0.31) mm] and LVESd [(7.98± 0.37) mm] and lowest EF [(42± 3.8)%] and FS [(40.3± 2.8)%]. The number of apoptotic myocardial cells was significant higher in ischemia group than control ( p&lt; 0 05), while it was significantly declined after treatment with promethazine ( p&lt; 0 05). Caspase-3 was significantly upregulated and Bcl-2 was downregulated in ischemia group which were all significantly reversed in drug group. Besides, Ki67 level was significantly reduced in ischemia group compared to control and higher in drug group than ischemia group, indicating that drug treatment increased cell proliferation ability. The levels of PI3K, GSK-3 and PKB in myocardial tissues were significantly declined in ischemia group and elevated after the treatment with promethazine without difference of PDK-1 level in myocardial tissues among the three groups. Conclusion: Promethazine inhibits apoptosis and promotes proliferation of myocardial cells in MIRI rats through PI3K/Akt signaling pathway.

Atorvastatin lipid nanocapsules and gold nanoparticles embedded in injectable thermo-gelling hydrogel scaffold containing adipose tissue extracellular matrix for myocardial tissue regeneration.

This study aimed to prepare, optimise, and characterise the novel hybrid hydrogel scaffold containing atorvastatin lipid nanocapsules (LNCs) and gold nanoparticles (NPs) to improve cardiomyoblasts proliferation and regeneration of myocardium. A thermo-responsive aminated guaran (AGG) hydrogel was prepared to encompass extracellular matrix (ECM) fetched from human adipose tissue. Emulsion phase-inversion technique was used to obtain LNCs. Biocompatibility, tensile strength, conductivity, and proliferation of human myocardial cells of the optimised formulation were studied. The LNCs have a spherical shape, and the optimised formulation showed a mean particle size of 18.79 nm, the zeta potential of - 11.4 mV, drug loading of 99.99%, and release efficiency percent over 72 h was 18.73%. The injectable thermo-sensitive hydrogel prepared using 1 w/v% of AGG, 35 w/w% of ECM, ∼0.5 mg/ml of gold NPs and atorvastatin loaded LNCs showed the best physical characteristics. The hybrid scaffold loaded with atorvastatin and gold NPs improved the proliferation of cardiomyoblasts more than sevenfold with enhanced cell attachment to the scaffold. The tensile strength and the conductivity of the scaffold were 300 kPa and 0.14 S/m, respectively. Injectable hybrid adipose tissue prepared by ECM and AGG hydrogel loaded with atorvastatin and gold NPs showed promising physical characteristics for myocardial tissue engineering.

LncRNA MEG3 accelerates apoptosis of hypoxic myocardial cells via FoxO1 signaling pathway.

To explore the effects of long non-coding ribonucleic acid (lncRNA) maternally expressed gene 3 (MEG3) on the proliferation and apoptosis of hypoxic myocardial cells by regulating the expression of forkhead box O1 (FoxO1). The myocardial A10 cell lines were divided into myocardial cell group (group A), hypoxic myocardial cell group (group B), and hypoxic myocardial cell + transfection with lncRNA MEG3 mimic group (group C). The correlations of the adenosine triphosphate (ATP) concentration, the degree of apoptosis, and the proliferation with FoxO1 and FoxO3a proteins in the cells were observed via ATP assay, Cell Counting Kit-8 (CCK-8) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, Western blotting, and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), respectively. The ATP concentration in myocardial cells was the highest in group A (p<0.05), and it was higher in group B than that in group C (p<0.05). The results of the CCK-8 assay showed that the proliferation rate of the myocardial cells was the highest in group A and the lowest in group C (p<0.05), and it was significantly increased in group B compared with that in group C (p<0.05). The results of the TUNEL assay revealed that the normal cells displayed the purple color, and apoptotic cells displayed the green color. The myocardial cells were arranged orderly, and the number of apoptotic cells was smaller in group A, the number of apoptotic cells was significantly larger in group B than that in group A, and it was the largest in group C (p<0.05). Moreover, the results of Western blotting manifested that the concentrations of FoxO1 and FoxO3 proteins in myocardial cells were the lowest in group A (p<0.05), and they were significantly higher in group C than those in group B (p<0.05). According to the results of qRT-PCR, the mRNA expressions of FoxO1 and FoxO3 in myocardial cells were the lowest in group A (p<0.05), and they were remarkably lower in group B than those in group C (p<0.05). LncRNA MEG3 can increase the activity of FoxO1 to promote myocardial apoptosis in a hypoxic environment.

Purification, characterization of Chondroitinase ABC from Sphingomonas paucimobilis and in vitro cardiocytoprotection of the enzymatically degraded CS-A

An extracellular chondroitinase ABC (ChSase ABC) produced by Sphingomonas paucimobilis was purified to homogeneity through ammonium sulfate precipitation, DEAE-Sepharose Fast Flow and Sephadex G-100 chromatography. The molecular weight was 82.3 kDa. It showed specific lyase activity toward chondroitin sulfate A (CS-A), CS-B, CS-C and hyaluronan (HA). Using CS-A as substrate, the specific activity was 98.04 U/mg, the maximal reaction rate (Vmax) and Michaelis-Menten constant (Km) were 0.49 μmol/min/ml and 0.79 mg/ml, respectively. Highest activity was obtained at pH 6.5 and 40 °C, and Hg2+ could strongly inhibit the enzyme activity. Mass spectrometry analysis indicated CS-A was degraded to unsaturated disaccharides by ChSase ABC. In vitro cytotoxic tests showed that CS-A oligosaccharide at the concentration of 50 and 100 μg/ml could promote the proliferation of normal H9c2 myocardial cells, decrease the damage induced by isoproterenol (ISO) and accelerate the recovery of cells injured by ISO. These findings suggested that ChSase ABC from Sphingomonas paucimobilis could be a promising tool for the structural analysis and bioactive oligosaccharide preparation of glucosaminoglycans.

Activation of Endogenous Cardiac Stem Cells by Apelin-13 in Infarcted Rat Heart.

Our previous study demonstrated that the apelin-APJ pathway contributed to myocardial regeneration and functional recovery after bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation during the differentiation of BM-MSCs into cardiomyogenic cells in acute myocardial infarction (AMI) rat models. However, the underlying mechanisms by which apelin promotes cardiac repair and functional recovery have not been completely clarified. In the present study, we investigated whether apelin could mobilize and activate endogenous cardiac stem cells and progenitors, thereby mediating regeneration and repair of the myocardium after AMI in rat models. Six-week-old male Sprague-Dawley rats underwent AMI and received apelin-13 (200 ng, n = 10) or an equivalent volume of saline by intramyocardial injection (n = 10); there was also a sham operation group (n = 8). Proliferation of endogenous cardiac stem cells was analyzed by immunofluorescence staining in rat infarcted myocardium, and heart function was evaluated by echocardiography at 28 days after apelin-13 injection. Treatment with apelin-13 led to a significant increase of Ki-67+-c-kit+/Sca-1+/Flk-1+ endogenous cardiac stem or progenitor cells in the border zone and infarct zone of rat hearts at 28 days after myocardial infarction (MI). Significant increases in the expression of c-kit, Sca-1, and Flk-1 on both levels of transcription and translation were confirmed by real-time polymerase chain reaction (RT-PCR) and Western blot. Treatment of apelin-13 also resulted in a significant reduction of infarct size and improvement of cardiac function post-MI. We conclude that apelin-13 is able to enhance mobilization, survival, and proliferation of endogenous myocardial stem cells in the injured heart, providing a novel mechanistic explanation for how apelin-13 might repair the heart and improve cardiac function. Thus, apelin-13 or pharmacological agonists of the APJ receptor could act as novel therapies for heart regeneration.

Stimulation of Proliferation and Differentiation of Rat Resident Myocardial Cells with Apoptotic Bodies of Cardiomyocytes.

Using the cell model of regenerative cardiomyogenesis (formation of contracting cardiomyocyte colonies from resident stem cells), we found that the addition of cardiomyocyte-derived apoptotic bodies to the culture of neonatal myocardial cells stimulated proliferation and differentiation of cardiomyocyte precursors and the frequency of their contraction was 1.5-fold higher than in the control. Systemic administration of cardiomyocyte-derived apoptotic bodies to Wistar rats with chronic postinfarction heart failure during the early period of myocardial remodeling considerably improved the contractile function of the heart.

Aquaporin in the proliferation and apoptosis of diabetic myocardial cells.

The aim of this study was to explore the effect of aquaporin on the molecular mechanism of human diabetic myocardial cell apoptosis. The methylthiazolyle tetrazolium assay was used to detect the inhibitory effect of different concentrations of aquaporin on cell growth. The rate of aquaporin-induced myocardial cell apoptosis was detected by flow cytometric analysis of Annexin V-fluorescein isothiocyanate/propidium iodide double-stained cells. We also attempted to quantify the expression of Bcl-2, Bax, caspase-3, and survivin in diabetic myocardial cells by western blot analysis. Aquaporin was found to inhibit the proliferation of diabetic myocardial cells in a concentration-dependent manner; the increase in aquaporin concentration led to an increase in Bax (apoptosis protein) expression, decrease in Bcl-2 expression (anti-apoptosis protein), increase in the Bax/Bcl-2 ratio, and a decrease in caspase-3 and survivin expression (P < 0.05). Therefore, aquaporin significantly inhibits the proliferation of diabetic myocardial cells and cell apoptosis in a dose-dependent manner by upregulating the ratio of Bax/Bcl-2 protein expression, activating the caspase-3 protein cascade, and regulating the expression of survivin.

Abstract 20451: Hedgehog Signaling and Cardiomyocyte Proliferation

Background: In contrast to adult mammalian heart, lower vertebrates such as newt exhibit a dramatic capacity for regeneration in response to injury. Understanding the underlying mechanisms and signaling pathways in newt could form the basis for regenerative therapies in mammals. In the present study, we explored the role of hedgehog (HH) signaling during cardiac regeneration. Methods and Results: To investigate cardiac regeneration (CR) in vivo, we performed ventricular resection studies in adult newt heart. Whole mount and histochemical studies revealed CR within 30 d of resection injury. Functional assessment by echocardiographic analysis showed improved ejection fraction from 22 ± 5% at 4 d to 42 ± 3% at 30 d of regeneration relative to control (60 ± 3%). Gene clustering and qRT-PCR analysis at 7d post injury indicated enrichment of hedgehog (HH) signaling factors including Shh and ptch1 by 3- and 2-fold respectively, suggesting a critical requirement of HH signaling at early stages of CR. We determined HH signaling is essential as pharmacological inhibition of the HH pathway resulted in complete ablation of CR. Using EdU-labeling and immunohistochemical analyses, we showed that HH signaling regulates proliferation of both epicardial and myocardial cells, as inhibition of the HH pathway reduced EdU-positive nuclei from 15 ± 2% to 5 ± 3%. In contrast, qRT-PCR analysis from murine hearts postnatal day (P) 2 to P14, showed reduced levels of HH signaling factors and cell-cycle associated mRNAs by 3.5-fold with concomitant increase of p21 by 2-fold. This implies HH signals are required for the proliferative process. Consistent with the newt studies, activation of HH in murine neonatal ventricular cardiomyocytes (NVCM) promoted cardiomyocyte proliferation, increasing positive nuclei from 10 ± 2% to 25 ± 3%, while inhibition of HH signaling reduced positivity to 6 ± 3%. qRT-PCR analysis established that activation of HH signaling in NVCM resulted in up regulation of transcripts associated with cardiomyocyte proliferation. Conclusion: These data indicated that HH signaling pathways modulate cardiomyocyte proliferation providing potential therapeutic targets for achieving mammalian cardiac regeneration.

Hyperglycemia Slows Embryonic Growth and Suppresses Cell Cycle via Cyclin D1 and p21

In pregnant women, the diabetic condition results in a three- to fivefold increased risk for fetal cardiac malformations as a result of elevated glucose concentrations and the resultant osmotic stress in the developing embryo and fetus. Heart development before septation in the chick embryo was studied under two hyperglycemic conditions. Pulsed hyperglycemia induced by daily administration of glucose during 3 days of development caused daily spikes in plasma glucose concentration. In a second model, sustained hyperglycemia was induced with a single injection of glucose into the yolk on day 0. The sustained model raised the average plasma glucose concentration from 70 mg/dL to 180 mg/dL and led to decreased gene expression of glucose transporter GLUT1. Both models of hyperglycemia reduced embryo size, increased mortality, and delayed development. Within the heart outflow tract, reduced proliferation of myocardial and endocardial cells resulted from the sustained hyperglycemia and hyperosmolarity. The cell cycle inhibitor p21 was significantly increased, whereas cyclin D1, a cell cycle promoter, decreased in sustained hyperglycemia compared with controls. The evidence suggests that hyperglycemia-induced developmental delays are associated with slowed cell cycle progression, leading to reduced cellular proliferation. The suppression of critical developmental steps may underlie the cardiac defects observed during late gestation under hyperglycemic conditions.

Vascular endothelial growth factor is crucial for erythropoietin-induced improvement of cardiac function in heart failure

We intended to delineate the mechanisms of erythropoietin (EPO)-induced cardiac vascular endothelial growth factor (VEGF) production and to establish if VEGF is crucial for EPO-induced improvement of cardiac performance. The effects of EPO on VEGF expression were studied in cultured cardiac cells and EPO-treated hearts. The role of VEGF in EPO-induced neovascularization was studied with two distinct VEGF-neutralizing antibodies or irrelevant control IgG in an aortic sprouting assay and in rats with heart failure (HF) after myocardial infarction (MI) treated with EPO. EPO-alfa (10 IU/mL) was used in vitro and darbepoetin alfa (40 microg/kg/3 weeks, starting 3 weeks after MI) in vivo. EPO stimulated VEGF mRNA expression through the signal transducers and activators of transcription-3 (STAT-3) pathway in neonatal rat cardiomyocytes, but not in endothelial cells or fibroblasts. Similarly, the direct effects of EPO on endothelial sprouting were modest and VEGF independent. In rats with HF, EPO increased VEGF protein expression predominantly in cardiomyocytes, associated with a 37% increase in capillary density and improved cardiac performance. Administration of VEGF-neutralizing antibodies abrogated the salutary effects of EPO on cardiac microvascularization and function. VEGF neutralization attenuated EPO-induced proliferation of myocardial endothelial cells and reduced myocardial incorporation of endothelial progenitor cells (EPCs) in rats with alkaline phosphatase-labelled bone marrow cells. VEGF is crucial for EPO-induced improvement of cardiac function in HF. EPO fosters VEGF expression predominantly in cardiomyocytes, which in turn stimulates myocardial endothelial proliferation and incorporation of EPCs.