Viability Of Cardiac Fibroblasts Research Articles

Single-cell sequencing reveals LRRc17-mediated modulation of cardiac fibroblast ferroptosis in regulating myocardial fibrosis after myocardial infarction

PurposeMyocardial fibrosis after myocardial infarction (MI) is one of the main causes of death in patients, but there is no effective treatment for myocardial fibrosis at present. Hence, it is important to elucidate the pathogenesis of fibrosis after MI and find therapeutic targets for regulating ventricular remodeling after MI. MethodsDifferentially expressed gene analysis, weighted Gene co-expression network analysis (WGCNA) and differential gene analysis in cardiac fibroblasts (CFs) were performed on the MI-related data (GSE153485 and GSE210159) from the GEO database to screen the hub genes related to ferroptosis in MI. After the establishment of MI model in vivo and in vitro, the myocardial CFs were observed by Masson staining, hematoxylin-eosin staining, CCK-8, and Transwell, and the changes of LRRc17 and ferroptosis-related proteins were detected by qRT-PCR and Western blotting. The expression of ROS was detected by fluorescence dye method. ResultsThree DEGs were identified in MI related to ferroptosis, among which LRRc17 was selected for subsequent study. In both in vitro and in vivo models of MI, we found a sustained downregulation of LRRc17 expression and the expression of ferroptosis-related proteins GPX-4 and xCT, but increased ROS expression and enhanced migration and viability of CFs. After oe-LRRc17 treatment, the expression levels of GPX-4 and xCT were restored, while ROS levels were inhibited, and the migration and viability of CFs were inhibited. After treatment with ferroptosis inducer Erastin, there were down-regulated expressions of GPX-4 and xCT, increased expression of ROS, and enhanced migration and viability of CFs. ConclusionLRRc17 affects ventricular remodeling by mediating ferroptosis in CFs to regulate the degree of fibrosis after MI.

MiR-486-5p diagnosed atrial fibrillation, predicted the risk of left atrial fibrosis, and regulated angiotensin II-induced cardiac fibrosis via modulating PI3K/Akt signaling through targeting FOXO1.

This study focused on miR-486-5p in atrial fibrillation (AF) evaluating its clinical significance and revealing its regulatory mechanism in cardiac fibroblasts, aiming to explore a novel biomarker for AF. The study enrolled 131 AF patients and 77 non-AF individuals. With the help of polymerase chain reaction (PCR), the expression of miR-486-5p was evaluated. The significance of miR-486-5p in the diagnosis of AF and the occurrence of left atrial fibrosis (LAF) was assessed by receiver operating curve (ROC) and logistic analyses. The regulatory effect and mechanism of miR-486-5p on cardiac fibrosis were investigated in human cardiac fibroblasts treated with angiotensin II. miR-486-5p was significantly upregulated in AF patients and discriminated AF patients from non-AF individuals. Increasing miR-486-5p showed a significant association with decreasing left ventricular ejection fraction (LVEF), increasing left atrial diameter (LAD) and left ventricular end-diastolic diameter (LVEDd), and the high incidence of LAF in AF patients. Moreover, miR-486-5p was identified as a risk factor for LAF and could distinguish AF patients with LAF and without LAF. In cardiac fibroblasts, angiotensin II induced the upregulation of miR-486-5p and promoted cell proliferation, migration, and collagen synthesis. miR-486-5p negatively regulated forkhead box O1 (FOXO1) and its knockdown could reverse the promoted effect of angiotensin II. FOXO1 alleviated the effect of miR-486-5p, and the miR-486-5p/FOXO1 could activate PI3K/Akt signaling. The activation of PI3K/Akt signaling alleviated the enhanced proliferation, migration, and collagen synthesis of cardiac fibroblasts induced by angiotensin II, and its inhibition showed opposite effects. Increased miR-486-5p served as a biomarker for the diagnosis and development prediction of AF. miR-486-5p regulated cardiac fibroblast viability and collagen synthesis via modulating the PI3K/Akt signaling through targeting FOXO1.

Long Noncoding RNA HLA Complex Group 18 Improves the Cell Proliferation of Myocardial Fibroblasts by Regulating the Hsa-microRNA-133a/Epidermal Growth Factor Receptor Axis.

Hsa-microRNA (has-miR)-133a inactivates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and suppresses the cell proliferation of myocardial fibroblasts by downregulation of the epidermal growth factor receptor (EGFR) expression. Bioinformatics analysis exhibits extended noncoding RNA HLA complex group 18 (lncRNA-HCG18) binds to hsa-miR-133a. The purpose of the current experiment is to explore whether lncRNA-HCG18 adsorbed hsa-miR-133a through sponging, resulting in decreased inhibition of hsa-miR-133a on EGFR and ultimately promoting the proliferation of myocardial fibroblasts. To verify and study the correlation and mechanism between lncRNA-HCG18, hsa-miR-133a, and their target genes. Firstly, after overexpression/silencing of lncRNA-HCG18 in myocardial fibroblasts, the level of hsa-miR-133a expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the EGFR, ERK1/2, and p-ERK1/2 expression levels were assessed by Western blotting to confirm that upregulation of EGFR and p-ERK1/2 protein levels by overexpression of lncRNA-HCG18, siRNA lncRNA-HCG18 (siHCG18) reduced the EGFR and p-ERK1/2 protein levels. Then, the luciferase reporter gene system was used to verify that lncRNA-HCG18 regulated EGFR expression by inhibiting the function of the hsa-miR-133a regulatory target gene. Then, a RAP assay was used to confirm that lncRNA-HCG18 interacted with hsa-miR-133a. Finally, the analysis of CCK-8 results indicated that the cell proliferation of myocardial fibroblasts was significantly reduced by siHCG18 while reversed by overexpression of lncRNA-HCG18. Thus, lncRNA-HCG18 inhibited cell viability of cardiac fibroblasts via the hsa-miR-133a/EGFR axis, which was regarded as a regulator of cell proliferation of cardiac fibroblasts in cardiovascular diseases.

Laser Technology for the Formation of Bioelectronic Nanocomposites Based on Single-Walled Carbon Nanotubes and Proteins with Different Structures, Electrical Conductivity and Biocompatibility

A laser technology for creating nanocomposites from alternating layers of albumin/collagen proteins with two types of single-walled carbon nanotubes (SWCNT) at concentrations of 0.001 and 0.01 wt.% was proposed. For this purpose, a setup with a diode laser (810 nm) and feedback system for controlling the temperature of the area affected by the radiation was developed. Raman spectroscopy was used to determine a decrease in the defectiveness of SWCNT with an increase in their concentration in the nanocomposite due to the formation of branched 3D networks with covalent bonds between nanotubes. It was revealed that adhesion of proteins to branched 3D networks from SWCNT occurred. The specific electrical conductivity of nanocomposites based on large SWCNT nanotubes was 3.2 and 4.3 S/m compared to that for nanocomposites based on small SWCNT with the same concentrations—1.1 and 1.8 S/m. An increase in the concentration and size of nanotubes provides higher porosity of nanocomposites. For small SWCNT-based nanocomposites, a significant number of mesopores up to 50 nm in size and the largest specific surface area and specific pore volume were found. Nanocomposites with small SWCNT (0.001 wt.%) provided the best cardiac fibroblast viability. Such technology can be potentially used to create bioelectronic components or scaffolds for heart tissue engineering.

Knockdown of ZFAS1 improved the cardiac function of myocardial infarction rats via regulating Wnt/β-catenin signaling pathway.

Myocardial infarction (MI) is a big health threat in the world, and it is characterized by high morbidity and mortality. However, current treatments are not effective enough, and novel therapeutic strategies need to be explored. ZFAS1 has been proved to be involved in the regulation of MI, but the specific mechanism remains unclear. MI rats were constructed through left anterior descending artery ligation, and hypoxia cell model was also established. The proliferation, invasion, and migration of cells were detected via CCK8, traswell, and wound healing methods. Immunohistochemistry staining, western blotting, and qRT-PCR were used to detect the levels of molecules. Knockdown of ZFAS1 significantly increased the proliferation, migration, and invasion of cardiac fibroblasts. Knockdown of ZFAS1 remarkably improved cardiac function via decreasing infarction ratio and increasing vWF expression, left ventricular ejection fraction, and left ventricular fractional shortening compared with group MI. Knockdown of ZFAS1 also suppressed Wnt/β-catenin pathway in vivo. The inhibition of Wnt/β-catenin remarkably reversed the influence of shZFAS1 on cardiac function and cardiac fibroblasts viability. Therefore, Knockdown of ZFAS1 could improve the cardiac function of myocardial infarction rats via regulating Wnt/β-catenin signaling pathway. The present study might provide new thoughts for the prevention and treatment of MI damage.

HIMF deletion ameliorates acute myocardial ischemic injury by promoting macrophage transformation to reparative subtype

Appropriately manipulating macrophage M1/M2 phenotypic transition is a promising therapeutic strategy for tissue repair after myocardial infarction (MI). Here we showed that gene ablation of hypoxia-induced mitogenic factor (HIMF) in mice (Himf−/− and HIMFflox/flox;Lyz2-Cre) attenuated M1 macrophage-dominated inflammatory response and promoted M2 macrophage accumulation in infarcted hearts. This in turn reduced myocardial infarct size and improved cardiac function after MI. Correspondingly, expression of HIMF in macrophages induced expression of pro-inflammatory cytokines; the culturing medium of HIMF-overexpressing macrophages impaired the cardiac fibroblast viability and function. Furthermore, macrophage HIMF was found to up-regulate C/EBP-homologous protein (CHOP) expression, which exaggerated the release of pro-inflammatory cytokines via activating signal transducer of activator of transcription 1 (STAT1) and 3 (STAT3) signaling. Together these data suggested that HIMF promotes M1-type and prohibits M2-type macrophage polarization by activating the CHOP–STAT1/STAT3 signaling pathway to negatively regulate myocardial repair. HIMF might thus constitute a novel target to treat MI.

Qingda granule attenuates cardiac fibrosis via suppression of the TGF-β1/Smad2/3 signaling pathway in vitro and in vivo

Cardiac fibrosis plays an important role in hypertension-related contractile dysfunction and heart failure. Qingda granule (QDG), derived from the Qingxuan Jiangya decoction, has been used clinically for more than 60 years to treat hypertension. However, the effect of QDG on hypertensive cardiac fibrosis remains largely unknown. The objective of this study was to investigate the effect of QDG on cardiac fibrosis and explore the underlying mechanism in vivo and in vitro. For in vivo experiments, 30 male spontaneously hypertensive rats were randomly divided into groups that received no QDG or one of three doses (0.45, 0.9 or 1.8 g/kg/day). Positive-control animals received valsartan (VAL, 7.2 mg/kg/day). Treatments were administered by gavage for 10 weeks. All three doses of QDG and VAL led to significantly lower blood pressure than in SHR animals. Besides, all three doses of QDG and VAL attenuated pathological changes in SHR animals. However, only intermediate, high concentrations of QDG and VAL led to significantly lower left ventricle ejection fraction and left ventricle fractional shortening than in SHR animals. Therefore, the minimum and effective QDG dose (intermediate concentration of QDG) was selected for subsequent animal experiments in this study. Our results showed that intermediate concentration of QDG also significantly mitigated the increases in levels of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), collagen III, transforming growth factor-β1 (TGF-β1) and in the ratio of phospho-Smad2/3 to total Smad2/3 protein in cardiac tissue, based on immunohistochemistry, Western blotting, and Masson staining. For in vitro experiments, primary cardiac fibroblasts were stimulated with 100 nM angiotensin II in the presence or absence of QDG. And we tested different concentrations of QDG (3.125, 6.25, 12.5, 25, 50 μg/mL) in the cell viability experiment. Our results showed that 3.125, 6.25 and 12.5 μg/mL of QDG treatment for 24 h didn’t affect the cell viability of cardiac fibroblasts. Consistently, QDG at 6.25 or 12.5 μg/mL significantly reduced cell viability and down-regulated α-SMA in primary cardiac fibroblasts were stimulated with 100 nM angiotensin II. Therefore, QDG at 12.5 μg/mL was chosen for the following cell experiment. Our results showed that QDG at 12.5 μg/mL alleviated the increase of PCNA, collagen Ⅲ, TGF-β1 expression, and the ratio of phospho-Smad2/3 to total Smad2/3 protein. Our studies in vitro and in vivo suggest that QDG reduces blood pressure and cardiac fibrosis as well as protecting cardiac function, and that it exerts these effects in part by suppressing TGF-β1/Smad2/3 signaling.

Xanthohumol Inhibits TGF-β1-Induced Cardiac Fibroblasts Activation via Mediating PTEN/Akt/mTOR Signaling Pathway.

BackgroundXanthohumol (Xn) is the most abundant prenylated flavonoid in Hops (Humulus lupulus L.), and exhibits a range of pharmacological activities. This study aimed to investigate the effect of Xn on TGF-β1-induced cardiac fibroblasts activation and elucidate the underlying mechanism.Materials and MethodsThe cellTiter 96® AQueous one solution cell proliferation assay kit was adopted to determine the cell viability of cardiac fibroblasts, and the proliferation was detected through 5-ethynyl-2ʹ-deoxyuridine (EdU) incorporation assay. The α-SMA protein expression was measured by using immunofluorescence and Western blotting. Western blotting was conducted to test the protein expressions of collagen I and III, PTEN, p-Akt, Akt, p-mTOR, mTOR, p-Smad3, Smad3 and GAPDH. The mRNA levels of α-SMA, collagen I and III were determined by quantitative real-time polymerase chain reaction (PCR).ResultsXn inhibited the TGF-β1-induced proliferation, differentiation and collagen overproduction of cardiac fibroblasts. TGF-β1 induced the down-regulated PTEN expression, Akt and mTOR phosphorylation. These effects of TGF-β1 were suppressed by Xn, while blocking of PTEN reduced Xn-mediated inhibitory effect on cardiac fibroblasts activation induced by TGF-β1.ConclusionXn inhibits TGF-β1-induced cardiac fibroblasts activation via mediating PTEN/Akt/mTOR signaling pathway.

MicroR-26b Targets High Mobility Group, AT-hook 2 to Ameliorate Myocardial Infarction-induced Fibrosis by Suppression of Cardiac Fibroblasts Activation.

Myocardial Fibrosis (MF) is an important physiological change after myocardial infarction (MI). MicroRNA-26b (MiR-26b) has a certain inhibitory effect on pulmonary fibrosis. However, the role of miR-26b in MI-induced MF rats and underlying molecular mechanisms remain unknown. Forty male Sprague Dawley (SD) rats weighing 200-250 g were divided into four groups (n=10): Sham group, MF group, MF + negative control (NC) agomir group and MF + miR-26b agomir group. Cardiac fibroblasts were isolated from cardiac tissue. Fibrosis levels were detected by MASSON staining, while the expression of related genes was detected by RT-qPCR, Western blotting and Immunohistochemistry, respectively. TargetScan and dual-luciferase reporter assay were utilized to predict the relationship between miR-26b and high mobility group, AT-hook 2 (HMGA2). The study found the expression of miR-26b to be down-regulated in the myocardium of MF rats (P<0.01). miR-26b overexpression in vitro significantly reduced the survival rate of cardiac fibroblasts and inhibited the expression of the fibrillar-associated protein (α-SMA alphasmooth muscle actin (α-SMA) and collagen I) (P<0.01). TargetScan indicated that HMGA2 was one of the target genes of miR-26b; dual-luciferase reporter assay further confirmed the targeted regulatory relationship (P<0.01). Moreover, miR-26b overexpression significantly reduced the expression of HMGA2 (P<0.01). Notably, HMGA2 overexpression reversed the inhibitory effect of miR-26b overexpression on cardiac fibroblast viability and the expression of α-SMA and collagen I (P<0.01). Animal experiments further indicated that miR-26b overexpression inhibited MIinduced rat MF by inhibiting the expression of HMGA2 (P<0.05, P<0.01). In short, these findings indicate that miR-26b targets HMGA2 to ameliorate MI-induced fibrosis by suppression of cardiac fibroblasts activation.

AB23A Reduces TGF-β1-Induced Human Cardiac Fibroblasts (HCF) Proliferation and Collagen Secretion via MER/ERK1/2/CREB Pathway

Background and Objectives: Myocardial fibrosis is associated with many forms of heart diseases which is characterized by the accumulation of activated cardiac fibroblasts (CFBs) and excess deposition of extracellular matrix (ECM). Natural compounds such as Alisol B 23-acetate has been proved to maintain the activation of ERK1/2, but whether it can affect cardiac fibroblasts by MER/ERK1/2/CREB signaling pathway is still unknown. Methods: The cell was identified with α-SMA protein level detected by immunofluorescence staining method. The cell proliferation was examined by CCK8 assay. Col I and Col III protein levels were examined by western blot and sirius red staining to detect the ECM level. Furthermore, p-MERK, MERK, P-ERK, ERK and CREB were examined by western blot to verify whether Alisol could activate the MERK/ERK1/2/CREB pathway in myocardial fibrosis. Results: CCK8 assay result indicated that Alisol reduced the cell viability of CFBs induced by TGF-β1. In addition, Alisol significantly decreased the ECM deposition of CFBs. Furthermore, Alisol could activate MERK/ERK1/2/CREB signaling pathway. Conclusion: These results verified that Alisol inhibited myocardial fibrosis via MERK/ERK1/2/CREB pathway.

The Association of Ascorbic Acid, Deferoxamine and N-Acetylcysteine Improves Cardiac Fibroblast Viability and Cellular Function Associated with Tissue Repair Damaged by Simulated Ischemia/Reperfusion.

Acute myocardial infarction is one of the leading causes of death worldwide and thus, an extensively studied disease. Nonetheless, the effects of ischemia/reperfusion injury elicited by oxidative stress on cardiac fibroblast function associated with tissue repair are not completely understood. Ascorbic acid, deferoxamine, and N-acetylcysteine (A/D/N) are antioxidants with known cardioprotective effects, but the potential beneficial effects of combining these antioxidants in the tissue repair properties of cardiac fibroblasts remain unknown. Thus, the aim of this study was to evaluate whether the pharmacological association of these antioxidants, at low concentrations, could confer protection to cardiac fibroblasts against simulated ischemia/reperfusion injury. To test this, neonatal rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion in the presence or absence of A/D/N treatment added at the beginning of simulated reperfusion. Cell viability was assessed using trypan blue staining, and intracellular reactive oxygen species (ROS) production was assessed using a 2′,7′-dichlorofluorescin diacetate probe. Cell death was measured by flow cytometry using propidium iodide. Cell signaling mechanisms, differentiation into myofibroblasts and pro-collagen I production were determined by Western blot, whereas migration was evaluated using the wound healing assay. Our results show that A/D/N association using a low concentration of each antioxidant increased cardiac fibroblast viability, but that their separate administration did not provide protection. In addition, A/D/N association attenuated oxidative stress triggered by simulated ischemia/reperfusion, induced phosphorylation of pro-survival extracellular-signal-regulated kinases 1/2 (ERK1/2) and PKB (protein kinase B)/Akt, and decreased phosphorylation of the pro-apoptotic proteins p38- mitogen-activated protein kinase (p38-MAPK) and c-Jun-N-terminal kinase (JNK). Moreover, treatment with A/D/N also reduced reperfusion-induced apoptosis, evidenced by a decrease in the sub-G1 population, lower fragmentation of pro-caspases 9 and 3, as well as increased B-cell lymphoma-extra large protein (Bcl-xL)/Bcl-2-associated X protein (Bax) ratio. Furthermore, simulated ischemia/reperfusion abolished serum-induced migration, TGF-β1 (transforming growth factor beta 1)-mediated cardiac fibroblast-to-cardiac myofibroblast differentiation, and angiotensin II-induced pro-collagen I synthesis, but these effects were prevented by treatment with A/D/N. In conclusion, this is the first study where a pharmacological combination of A/D/N, at low concentrations, protected cardiac fibroblast viability and function after simulated ischemia/reperfusion, and thereby represents a novel therapeutic approach for cardioprotection.

Analyis of biological compatibility of polylactide nanofibrous matrix vitalized with cardiac fibroblasts in a porcine model

Currently the use of synthetic biodegradable polymers based on polyurethane, polycaprolactone, polylactic and polyglycolic acids structures and their co-polymers is one of the most perspective directions of tissue engineering development. Electrospinning was found as an optimal way to produce nanofibers suitable for building several types of biomaterial scaffolds that are used in cell therapy. This technology allows creating a stable biodegradable and highly biocompatible matrix. In this study we investigate the viability of cardiac fibroblasts cultivated on polymeric nanofibrous scaffolds in vitro and in vivo after implantation in the myocardium of an experimental animal. Polymeric nanofibers were produced on an electrospinning unit. Prepared matrixes were vitalized with cell cultures, received from atriums of several mini-pigs. Cell viability was estimated by the use of XTT based colorimetric assay. Two groups of mini-pigs were selected for this research. The first group underwent a procedure of intramyocardial implantation of a matrix, grown with cardiac cell culture. In the second group a clear polymeric matrix was implanted. Seven days after the procedure animals were sacrificed and fragments of myocardium containing implants were harvested. Frozen sections were prepared immediately, then a standard histological analysis and immunofluorescent staining were performed. Current results can be significant for further development of polymeric scaffolds and for research of biophysical and electrophysiological features of cardiac cell cultures, what will help to expand the abilities of contemporary regenerative medicine and may become a standard of autological biological therapy.

MicroRNA-33a deficiency inhibits proliferation and fibrosis through inactivation of TGF-β/Smad pathway in human cardiac fibroblasts.

Cardiac fibroblasts (CFBs) play pivotal roles in myocardial fibrosis, which is the leading cause of arrhythmia. This study was aimed to investigate the modulation of microRNA (miR)-33a on proliferation, apoptosis and fibrosis of human CFBs. CFBs were respectively transfected with miR-control, miR-33a mimic or miR-33a inhibitor, followed by induction of transforming growth factor-β (TGF-β). Non-treated CFBs acted as control. Cell viability, apoptosis, and fibrosis which reflected by expressions of Col-I, Col-III and α-smooth muscle actin (α-SMA) were evaluated by CCK-8 assay, flow cytometry, qRT-PCR and Western blot analysis. Finally, key kinases involved in the TGF-β/Smad pathway were evaluated by Western blot analysis. TGF-β enhanced CFB viability, and expression levels of Col-I, Col-III and α-SMA in CFBs (P < 0.01 or P < 0.001). The increased CFB proliferation, and upregulation of Col-I, Col-III and α-SMA were all further enhanced by miR-33a mimic (P < 0.05 or P < 0.001), whereas reversed by miR-33a inhibitor (P < 0.05, P < 0.01 or P < 0.001). The CFB apoptosis was remarkably promoted by miR-33a inhibitor (P < 0.001). Results of signaling pathway showed that phosphorylated levels of Smad-2 and Smad-3 were both upregulated by TGF-β (P < 0.001). The upregulated phosphorylations were further improved by miR-33 mimic (P < 0.05) while reversed by miR-33a inhibitor (P < 0.05 or P < 0.001). miR-33a deficiency inhibits proliferation and fibrosis of CFBs while promotes CFB apoptosis by inactivation of TGF-β/Smad pathway.

Combined hypoxia and sodium nitrite pretreatment for cardiomyocyte protection in vitro.

Methods that increase cardiomyocyte survival upon exposure to ischemia, hypoxia and reoxygenation injuries are required to improve the efficacy of cardiac cell therapy and enhance the viability and function of engineered tissues. We investigated the effect of combined hypoxia/NaNO2 pretreatment on rat neonatal cardiomyocyte (CM), cardiac fibroblast, and human embryonic stem cell-derived CM (hESC-CM) survival upon exposure to hypoxia/reoxygenation (H/R) injury in vitro. Cells were pretreated with and without hypoxia and/or various concentrations of NaNO2 for 20 min, then incubated for 2 h under hypoxic conditions, followed by 2 h in normoxia. The control cells were maintained under normoxia for 4 h. Pretreatment with either hypoxia or NaNO2 significantly increased CM viability but had no effect on cardiac fibroblast viability. Combined hypoxia/NaNO2 pretreatment significantly increased CM viability but significantly decreased cardiac fibroblast viability. In rat neonatal CMs, cell death, as determined by lactate dehydrogenase (LDH) activity, was significantly reduced with hypoxia/NaNO2 pretreatment; and in hESC-CMs, hypoxia/NaNO2 pretreatment increased the BCL-2/BAX gene expression ratio, suggesting that hypoxia/NaNO2 pretreatment promotes cell viability by downregulating apoptosis. Additionally, we found a correlation between the prosurvival effect of hypoxia/NaNO2 pretreatment and the myoglobin content of the cells by comparing neonatal rat ventricular and atrial CMs, which express high and low myoglobin respectively. Functionally, hypoxia/NaNO2 pretreatment significantly improved the excitation threshold upon H/R injury to the level observed for uninjured cells, whereas pretreatment did not affect the maximum capture rate. Hence, hypoxia/NaNO2 pretreatment may serve as a strategy to increase CM survival in cardiac regenerative therapy applications and tissue engineering.

Epigallocatechin Gallate Attenuated the Activation of Rat Cardiac Fibroblasts Induced by Angiotensin II via Regulating β-Arrestin1

Background/Aims: Angiotensin II (AngII) activated cardiac fibroblasts (CFs) predominantly through AngII subtype 1a receptor (AT1aR). This study was carried out to explore the potential inhibitory effects and mechanisms of epigallocatechin gallate (EGCG) on AngII induced rat CFs. Methods: Viability, proliferation and collagen production of CFs were measured by MTT assay, [³H]-thymidine and [³H]-proline incorporation respectively. β-arrestin1 (βarr1), AT1aR and AT1bR mRNA levels were determined by quantitative PCR. AT1R, Gq, βarr 1/2, phosphorylated kinase C (p-PKC)-delta expressions were detected by western blotting. We blocked βarr1 expression using βarr1 small interfering RNA (siRNA). Results: EGCG inhibited the activation of CFs induced by AngII. βarr1 mRNA level revealed a positive correlation with the viability of CFs. SiRNA targeting βarr1 blocked the activation of CFs. In vitro, AngII increased βarr1 mRNA, total and membrane βarr1 protein expressions, but reduced AT1aR mRNA, global and membrane AT1R, total Gq and cytoplasmic p-PKC-delta levels. Administration of EGCG restored the above abnormalities, whereas Gq levels were not affected. Conclusion: Our findings showed that βarr1 is essential for AngII-mediated activation of CFs. EGCG attenuated CFs activation induced by AngII via regulating βarr1 and thus, modulating AT1aR mediated signaling.

Effects of propofol on the expression of matric metalloproteinases in rat cardiac fibroblasts after hypoxia and reoxygenation

Propofol is known to protect the myocardium against ischaemia/reperfusion (I/R) injury through its antioxidant and anti-inflammatory properties. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved in cell migration and invasion, and mediate tissue remodelling during I/R injury. They are regulated by various mechanisms including oxidative stress and AKT and ERK pathways. We investigated whether propofol affected the expression of MMPs and subsequent cell migration and invasion and the signalling pathways involved in primary rat cardiac fibroblasts undergoing hypoxia and reoxygenation. The phosphorylation of ERK and AKT signalling pathways was examined by western blot analysis in rat primary cardiac fibroblasts after hypoxia and reoxygenation. mRNA expression of MMP and TIMPS was analysed by real-time PCR, and proteolytic activities of MMP-2 and -9 were assessed. The effects of propofol on migration, invasion, wound healing, and cell proliferation activity were evaluated after reoxygenation. Propofol induced AKT and ERK1/2 activation. Subsequent activation of MMPs resulted in increased cell migration, invasion, and wound-healing activity under hypoxia-reoxygenation, which was decreased by LY294002 (AKT inhibitor) and U0126 (ERK inhibitor) in rat cardiac fibroblasts. However, propofol had no effect on proliferation or viability of cardiac fibroblasts after hypoxia-reoxygenation. Propofol affected the expression of MMPs and TIMPs and subsequently induced cell migration and invasive ability, through activation of the ERK and AKT signalling pathway in hypoxia-reoxygenated rat cardiac fibroblasts.

NF-κB inhibition compromises cardiac fibroblast viability under hypoxia

Cardiac fibroblasts are reported to be relatively resistant to stress stimuli compared to cardiac myocytes and fibroblasts of non-cardiac origin. However, the mechanisms that facilitate their survival under conditions of stress remain unclear. We explored the possibility that NF-κB protects cardiac fibroblasts from hypoxia-induced cell death. Further, we examined the expression of the antiapoptotic cIAP-2 and Bcl-2 in hypoxic cardiac fibroblasts, and their possible regulation by NF-κB. Phase contrast microscopy and propidium iodide staining revealed that cardiac fibroblasts are more resistant than pulmonary fibroblasts to hypoxia. Electrophoretic Mobility Shift Assay showed that hypoxia activates NF-κB in cardiac fibroblasts. Supershift assay indicated that the active NF-κB complex is a p65/p50 heterodimer. An I-κB-super-repressor was constructed that prevented NF-κB activation and compromised cell viability under hypoxic but not normoxic conditions. Similar results were obtained with Bay 11-7085, an inhibitor of NF-κB. Western blot analysis showed constitutive levels of Bcl-2 and hypoxic induction of cIAP-2 in these cells. NF-κB inhibition reduced cIAP-2 but not Bcl-2 levels in hypoxic cardiac fibroblasts. The results show for the first time that NF-κB is an important effector of survival in cardiac fibroblasts under hypoxic stress and that regulation of cIAP-2 expression may contribute to its pro-survival role.

Abstract 1419: Biocompatible 3-Dimensional RGD-Modified Alginate Scaffold Designed to Enhance Cell Transplantation to Infarcted Myocardium

Introduction Cell transplantation strategies for cardiac repair are currently limited by poor survival of transplanted cells. Three dimensional scaffolds improve cell transplant survival after delivery to infarcted myocardium. We aimed to design a biocompatible porous 3D scaffold using custom purified alginate that augments cell viability. Two-dimensional surfaces coated with cyclic RGDfK peptides have been shown to improve cell viability. We hypothesized that covalent attachment of cRGDfK peptide to an alginate scaffold would improve cell viability in 3D. Methods Cyclic RGDfK peptide modified porous scaffolds were fabricated by freeze gelation and subsequently seeded with 3x10e6 neonatal rat cardiomyocytes or 3x10e6 neonatal rat cardiac fibroblasts. Cell containing scaffolds were cultured for one week and cell viability was determined by trypan blue exclusion. Scaffolds were implanted intramuscularly in rats (n=4) for 1 month to determine biocompatibility by multinucleated giant cell formation, compared to immunogenic suture material. Efficacy of circular scaffold (16 mm x 0.75 mm) application to the epicardial surface of rat hearts (n=12) was evaluated macroscopically and by ED-2 positive macrophage staining 1 week after transplantation. Results Scaffolds with interconnected pores (100–200 μm) allowed seeding and even distribution of cells. At 1 week, neonatal rat myocyte viability inside scaffolds increased from 3.3±1.2% (0 mg/g cRGDfK) to 12.3±0.1% (10 mg/g cRGDfk) to 28.9±7.3% (10 mg/g cRGDfk + gelatin) (P&lt;0.05). Clusters of beating myocytes could be detected. Neonatal rat cardiac fibroblast viability increased from 48.8±21% (0 mg/g cRGDfK) to 77.2±3.2% (10 mg/g cRGDfK) (P&lt;0.05). One month after intramuscular scaffold implantation, 1.2±0.7 multinucleated cells could be seen per HPF throughout the scaffold, vs 17.5±4.2 around immunogenic sutures (P&lt;0.05). Scaffolds integrated with the epicardial surface 1 week after transplantation. ED-2 staining was negative. Conclusion Three dimensional scaffolds generated from custom purified alginate modified with cRGDfK peptide promote cardiac cell viability in vitro. Scaffolds are biocompatible and may be used to transplant cells to the myocardium by epicardial application.

Angiotensin II modulates nitric oxide-induced cardiac fibroblast apoptosis by activation of AKT/PKB.

Previously we found that interleukin-1beta (IL-1beta)-activated inducible nitric oxide (NO) synthase (iNOS) expression and that NO production can trigger cardiac fibroblast (CFb) apoptosis. Here, we provide evidence that angiotensin II (ANG II) significantly attenuated IL-1beta-induced iNOS expression and NO production in CFbs while simultaneously decreasing apoptotic frequency. The anti-apoptotic effect of ANG II was abolished when cells were pretreated with the specific ANG II type 1 receptor (AT1) antagonist losartan, but not by the AT2 antagonist DP-123319. Furthermore, ANG II also protected CFbs from apoptosis induced by the NO donor diethylenetriamine NONOate and this effect was associated with phosphorylation of Akt/protein kinase B at Ser473. The effects of ANG II on Akt phosphorylation and NO donor-induced CFb apoptosis were abrogated when cells were preincubated with the specific phosphatidylinositol 3-kinase inhibitors wortmannin or LY-294002. These data demonstrate that ANG II protection of CFbs from IL-1beta-induced apoptosis is associated with downregulation of iNOS expression and requires an intact phosphatidylinositol 3-kinase-Akt survival signal pathway. The findings suggest that ANG II and NO may play a role in regulating the cell population size by their countervailing influences on cardiac fibroblast viability.

Insulin-like growth factor I retards apoptotic signaling induced by ethanol in cardiomyocytes

Apoptosis of cardiac muscle cells may contribute to the development of cardiomyopathy and heart failure. Alcohol (ethanol) abuse is a major cause of cardiomyopathy, but its underlying mechanism remains unknown. To determine whether ethanol causes apoptosis in cardiac muscle and whether insulin-like growth factor I (IGF I) improves cardiac muscle survival upon ethanol exposure, we have defined the effects of ethanol and IGF I in primary cardiomyocytes. Ethanol decreased cell viability in dose-response manner from 0.2% to 1%. In contrast, ethanol (0.2–1%) did not alter viability of cardiac fibroblasts. To assess the occurrence of apoptosis, DNA fragmentation was determined with quantitation of nucleosomes. Nucleosomes were increased in ethanol-treated cells, thus confirming the apoptotic effects of ethanol. The pro-apoptotic Bax protein and Caspase 3 are important proteins of apoptotic signaling. The content of Bax and the activities of Caspase 3 were increased upon ethanol exposure. IGF I partially suppressed Bax induction, Caspase 3 activation, DNA fragmentation, and increased cardiomyocyte survival. The effects of IGF I on ethanol-induced apoptosis can be inhibited with a chemical inhibitor of PI 3 Kinase (LY-294002), suggesting that anti-apoptotic actions of IGF I involves PI 3 Kinase. These results may have important implications on further understanding the pathogenesis of alcoholic heart disease and the development of new strategies to treat alcoholic cardiomyopathy.