[Metabolic mechanism and intervention strategy of atrial fibrillation in the elderly].

Atrial fibrillation (AF) is a cardiovascular epidemic that occurs primarily in the elderly with primary cardiovascular diseases, leading to severe consequences such as stroke and heart failure. The heart is an energy-consuming organ, which requires a high degree of metabolic flexibility to ensure a quick switch of metabolic substrates to meet its energy needs in response to physiological and pathological stimulation. Metabolism is closely related to the occurrence of AF, and AF patients manifest metabolic inflexibility, such as insulin resistance and the metabolic shift from aerobic metabolism to anaerobic glycolysis. Moreover, our research group and the others have shown that metabolic inflexibility is a crucial pathologic mechanism for AF. Energy metabolism is closely linked to the aging process and aging-related diseases, and impaired metabolic flexibility is considered as an essential driver of aging. Therefore, this review focuses on the alteration of metabolic flexibility in the elderly and reveals that impaired metabolic flexibility may be an important driver for the high prevalence of AF in the elderly, hoping to provide intervention strategies for the prevention and treatment of AF in the elderly.

[Active components and action mechanism of Shenmai Injection in treatment of atrial fibrillation based on network pharmacology and molecular docking].

This study aims to explore the active components and molecular mechanism of Shenmai Injection in the treatment of atrial fibrillation(AF) based on the application of network pharmacology and molecular docking technology. The chemical components of single herbs of Shenmai Injection were collected from TCMSP and TCMID, with the standard chemical name and PubChem CID(referred to as CID) obtained from PubChem database. The active components were screened using SwissADME, and their targets were predicted using SwissTargetPrediction. Targets related to AF treatment were identified using GeneCards, OMIM, and other databases. Venn diagram was constructed using Venny 2.1 to obtain the intersection targets. The single herb-active component-potential target network was constructed using Cytoscape, and the clusterProfiler R function package was used to perform the gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment. The protein-protein interaction(PPI) network of intersection targets was generated based on the STRING database. The hub target protein was identified by visualization using Cytoscape, and then docked to its reverse-selected active components. The analysis showed that there were 65 active components with 681 corresponding targets in Shenmai Injection, 2 798 targets related to AF treatment, and 235 intersection targets involving 2 549 GO functions and 153 KEGG pathways. Finally, hub target proteins, including RAC-alpha serine/threonine-protein kinase(AKT1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3 CA), and estrogen receptor 1(ESR1), were screened out by PPI network visualization. The molecular docking was performed for 39 active components screened out in reverse, among which 30 active components de-monstrated high affinity. Among them, homoisoflavanoids CID 10871974, CID 5319742, and CID 10361149 had stronger affinity docking with AKT1. This study preliminarily indicates that Shenmai Injection treats AF through multiple components, multiple targets, and multiple pathways. Homoisoflavonoids of Ophiopogon japonicus are its important active components, which target AKT1 to regulate metabolism, inflammation, and apoptosis in AF treatment.

Anti-arrhythmic and anti-heart failure effects of low-level electrical stimulation on aortic root ventricular ganglionated plexi.

BACKGROUND: It remains uncertain whether low-level electrical stimulation (LL-ES) of the ventricular ganglionated plexi (GP) improves heart function. This study investigated the anti-arrhythmic and anti-heart failure effects of LL-ES of the aortic root ventricular GP (ARVGP). METHODS: Thirty dogs were divided randomly into control, drug, and LL-ES groups after performing rapid right ventricular pacing to establish a heart failure (HF) model. The inducing rate of arrhythmia; levels of bioactive factors influencing HF, including angiotensin II type I receptor (AT-1R), transforming growth factor-beta (TGF-ß), matrix metalloproteinase (MMP), and phosphorylated extracellular signal-regulated kinase (p-ERK1/2); left ventricular stroke volume (LVSV), and left ventricular ejection fraction (LVEF)were measured after treatment with placebo, drugs, and LL-ES. RESULTS: The inducing rate of atrial arrhythmia decreased from 60% in the control group to 50% in the drug group and 10% in the LL-ES group (p = .033 vs. drug group) after 1 week of treatment. The ventricular effective refractory period was prolonged from 139 ± 8 ms in the drug group to 166 ± 13 ms in the LL-ES group (p = .001). Compared to the drug group, the expressions of AT-1R, TGF-ß, and MMP proteins were down-regulated in the LL-ES group, whereas that of p-ERK1/2 was significantly increased (all p = .001). Moreover, in the LL-ES group, LVSV increased markedly from 13.16 ± 0.22 to 16.86 ± 0.27 mL, relative to that in the drug group (p = .001), and LVEF increased significantly from 38.48% ± 0.53% to 48.94% ± 0.57% during the same time frame (p = .001). CONCLUSION: Short-term LL-ES of ARVGP had both anti-arrhythmic and anti-inflammatory effects and contributed to the treatment of tachycardia-induced HF and its associated arrhythmia.

Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

OBJECTIVES: Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. MATERIALS AND METHODS: Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. RESULTS: Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although ß-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on α-AR signalling than ß-AR signalling. In addition, selective activation of α1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α2 - or ß-AR signalling induced no or less differentiation, respectively. EPI- and α1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. CONCLUSIONS: These results demonstrate that activation of ARs, particularly of α1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling.

Correlating blood levels of 8-hydroxydeoxyguanosine to hOGG1 genotypes and the incidence of ischemic cardiomyopathy.

We measured the serum levels of 8-hydroxydeoxyguanosine (8-OHdG) and investigated whether these levels correlate with incidence of ischemic cardiomyopathy (ICM), and whether these levels correlate with underlying oxidative stress in patients with ICM. Polymerase chain reaction-restriction fragment length polymorphism analysis was performed to assess the prevalence of the Ser/Cys polymorphism in the human 8-oxoguanine glycosylase (hOGG1) gene. We analyzed the samples from 246 ICM cases (the ICM group) and another 246 age- and sex-matched volunteers with normal coronary artery function (the control group). Levels of 8-OHdG in participants' blood samples were 6.7 ± 1.7 and 3.0 ± 0.8 in the ICM and control groups, respectively (p < 0.05). Although there were no differences in allele frequency (p = 0.140), significant differences were present in the genotype distributions (p = 0.002). The Cys/Cys genotype correlated strongly with the risk of developing ICM (odds ratio, 2.2; 95% confidence interval, 1.4-3.3). Treating the Ser/Ser and Ser/Cys genotypes as members of the same group increased the predicted ICM risk for patients carrying the Cys/Cys genotype (odds ratio, 1.9; 95% confidence interval, 1.2-2.9). The serum level of 8-OHdG in the ICM group was higher than that in the control group (p < 0.05) and significantly increased in those carrying the Cys/Cys genotype (8.7 ± 1.7 for the Cys/Cys group, and 4.5 ± 0.8 for the Ser/Ser+Ser/Cys group; p < 0.05). Patients carrying the Cys/Cys genotype had a significantly increased risk of developing ICM. Serum levels of 8-OHdG were significantly increased in patients with ICM.

Cilostazol suppresses angiotensin II-induced apoptosis in endothelial cells.

Patients with essential hypertension undergo endothelial dysfunction, particularly in the conduit arteries. Cilostazol, a type III phosphodiesterase inhibitor, serves a role in the inhibition of platelet aggregation and it is widely used in the treatment of peripheral vascular diseases. Previous studies have suggested that cilostazol suppresses endothelial dysfunction; however, it remains unknown whether cilostazol protects the endothelial function in essential hypertension. The aim of the present study was to investigate whether, and how, cilostazol suppresses angiotensin II (angII)­induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) and Sprague Dawley rats were exposed to angII and treated with cilostazol. Endothelial cell apoptosis and function, nitric oxide and superoxide production, phosphorylation (p) of Akt, and caspase­3 protein expression levels were investigated. AngII exposure resulted in the apoptosis of endothelial cells in vitro and in vivo. In vitro, cilostazol significantly suppressed the angII­induced apoptosis of HUVECs; however, this effect was reduced in the presence of LY294002, a phosphoinositide 3 kinase (PI3K) inhibitor. Furthermore, cilostazol suppressed the angII­induced p­Akt downregulation and cleaved caspase­3 upregulation. These effects were also alleviated by LY294002. In vivo, cilostazol suppressed the angII­induced endothelial cell apoptosis and dysfunction. Cilostazol was also demonstrated to partially reduced the angII­induced increase in superoxide production. The results of the present study suggested that cilostazol suppresses endothelial apoptosis and dysfunction by modulating the PI3K/Akt pathway.

Three-dimensional poly-(ε-caprolactone) nanofibrous scaffolds directly promote the cardiomyocyte differentiation of murine-induced pluripotent stem cells through Wnt/ß-catenin signaling.

BACKGROUND: Environmental factors are important for stem cell lineage specification, and increasing evidence indicates that the nanoscale geometry/topography of the extracellular matrix (ECM) directs stem cell fate. Recently, many three-dimensional (3D) biomimetic nanofibrous scaffolds resembling many characteristics of the native ECM have been used in stem cell-based myocardial tissue engineering. However, the biophysical role and underlying mechanism of 3D nanofibrous scaffolds in cardiomyocyte differentiation of induced pluripotent stem cells (iPSCs) remain unclear. RESULTS: Here, we fabricated a 3D poly-(ε-caprolactone) (PCL) nanofibrous scaffold using the electrospinning method and verified its nanotopography and porous structure by scanning electron microscopy. We seeded murine iPSCs (miPSCs) directly on the 3D PCL nanofibrous scaffold and initiated non-directed, spontaneous differentiation using the monolayer method. After the 3D PCL nanofibrous scaffold was gelatin coated, it was suitable for monolayer miPSC cultivation and cardiomyocyte differentiation. At day 15 of differentiation, miPSCs differentiated into functional cardiomyocytes on the 3D PCL nanofibrous scaffold as evidenced by positive immunostaining of cardiac-specific proteins including cardiac troponin T (cTnT) and myosin light chain 2a (MLC2a). In addition, flow cytometric analysis of cTnT-positive cells and cardiac-specific gene and protein expression of cTnT and sarcomeric alpha actinin (α-actinin) demonstrated that the cardiomyocyte differentiation of miPSCs was more efficient on the 3D PCL nanofibrous scaffold than on normal tissue culture plates (TCPs). Furthermore, early inhibition of Wnt/ß-catenin signaling by the selective antagonist Dickkopf-1 significantly reduced the activity of Wnt/ß-catenin signaling and decreased the cardiomyocyte differentiation of miPSCs cultured on the 3D PCL nanofibrous scaffold, while the early activation of Wnt/ß-catenin signaling by CHIR99021 further increased the cardiomyocyte differentiation of miPSCs. CONCLUSION: These results indicated that the electrospun 3D PCL nanofibrous scaffolds directly promoted the cardiomyocyte differentiation of miPSCs, which was mediated by the activation of the Wnt/ß-catenin signaling during the early period of differentiation. These findings highlighted the biophysical role of 3D nanofibrous scaffolds during the cardiomyocyte differentiation of miPSCs and revealed its underlying mechanism involving Wnt/ß-catenin signaling, which will be helpful in guiding future stem cell- and scaffold-based myocardium bioengineering.

Low-Level Electrical Stimulation of Aortic Root Ventricular Ganglionated Plexi Attenuates Autonomic Nervous System-Mediated Atrial Fibrillation.

OBJECTIVES: This study investigated the effect of electrical stimulation of aortic root ventricular ganglionated plexi (GP) on atrial fibrillation (AF) inducibility. BACKGROUND: The ventricular GP are interconnected with atrial GP to govern heart function, although the effect of ventricular GP modification on control of AF remains unknown. METHODS: Effective refractory periods (ERPs) of test pulmonary veins (PVs) were measured at baseline and during high-level (HL-ES) and low-level (LL-ES) electrical stimulation of the aortic root GP. The arrhythmogenic threshold of acetylcholine and isoproterenol was determined at baseline and during HL-ES and LL-ES. Moreover, AF was induced at PVs by programmed electrical stimulation after HL-ES or LL-ES. Immunohistochemistry staining was performed to examine the autonomic activity from aortic root GP to the PVs. RESULTS: Compared with the baseline group, HL-ES of aortic root GP significantly shortened atrial ERP (95 ± 13 ms vs. 122 ± 9 ms) and PV ERP (104 ± 11 ms vs. 131 ± 12 ms); decreased the threshold concentration of AF by both acetylcholine (1.3 ± 0.2 µmol/l vs. 3.2 ± 0.3 µmol/l) and isoproterenol (0.3 ± 0.1 µmol/l vs. 1.3 ± 0.2 µmol/l); and increased the AF-inducing rate from PVs (90% vs. 30%). In contrast, LL-ES of the GP prevented the shortening of ERP and PV ERP to 125 ± 10 ms and 133 ± 11 ms, respectively; increased threshold levels of acetylcholine and isoproterenol to 5.7 ± 0.4 µmol/l and 3.2 ± 0.3 µmol/l; and decreased the AF-inducing rate to 5%. We also found that the biotinylated dextran amine-containing varicose fibers projected directly from the aortic root GP to the left PVs. CONCLUSIONS: These findings suggest that autonomic innervations of left PVs partly originated from aortic root ventricular GP. Moreover, LL-ES of aortic root ventricular GP suppressed AF inducibility and arose from PVs mediated by the autonomic nervous system.

Validation of the Andon KD-5917 automatic upper arm blood pressure monitor, for clinic use and self-measurement, according to the European Society of Hypertension International Protocol revision 2010.

OBJECTIVE: To validate the Andon KD-5917 automatic upper arm blood pressure monitor according to the European Society of Hypertension International Protocol revision 2010. MATERIALS AND METHODS: Sequential same-left-arm measurements of systolic blood pressure (SBP) and diastolic blood pressure (DBP) were obtained in 33 participants using the mercury sphygmomanometer and the test device. According to the validation protocol, 99 pairs of test device and reference blood pressure measurements (three pairs for each of the 33 participants) were obtained in the study. RESULTS: The device produced 73, 98, and 99 measurements within 5, 10, and 15 mmHg for SBP and 86, 98, and 99 for DBP, respectively. The mean ± SD device-observer difference was 3.07 ± 3.68 mmHg for SBP and -0.89 ± 3.72 mmHg for DBP. The number of patients with two or three of the device-observer difference within 5 mmHg was 26 for SBP and 29 for DBP, and no patient had a device-observer difference within 5 mmHg. CONCLUSION: The Andon KD-5917 automatic upper arm blood pressure monitor can be recommended for clinical use and self-measurement in an adult population on the basis of the European Society of Hypertension International Protocol revision 2010.

The independent role of the aortic root ganglionated plexi in the initiation of atrial fibrillation: An experimental study.

OBJECTIVE: The major atrial ganglionated plexi (GP) can initiate atrial fibrillation alone without any contribution from the extrinsic cardiac nervous system. However, if stimulation of the ventricular GP, especially the aortic root GP, can provoke atrial fibrillation (AF) alone is unknown. Our study was designed to investigate the independent role of aortic root GP activity in the initiation of AF. METHODS: In 10 Langendorff-perfused canine hearts, the atrial effective refractory period, pulmonary vein effective refractory period, and percentage of AF induced were measured at baseline and during aortic root GP stimulation. RESULTS: Stimulation of the aortic root GP shortened the atrial effective refractory period from 128 ± 10 ms at baseline to 103 ± 15 ms (P < .05) and shortened the pulmonary vein effective refractory period from 139 ± 14 ms to 114 ± 15 ms (P < .05). Furthermore, the percentage of AF induced in the 10 isolated hearts increased from 10% at baseline to 90% during aortic root GP stimulation (P < .05). CONCLUSIONS: In Langendorff-perfused canine hearts, stimulation of the aortic root GP provokes AF in the absence of any extrinsic cardiac nerve activity. The aortic root GP is an important element in the intrinsic neuronal loop that can increase the risk of AF in isolated heart models.

The long-term differentiation of embryonic stem cells into cardiomyocytes: an indirect co-culture model.

BACKGROUND: Embryonic Stem Cells (ESCs) can differentiate into cardiomyocytes (CMs) in vitro but the differentiation level from ESCs is low. Here we describe a simple co-culture model by commercially available Millicell™ hanging cell culture inserts to control the long-term differentiation of ESCs into CMs. METHODOLOGY/PRINCIPAL FINDINGS: Mouse ESCs were cultured in hanging drops to form embryoid bodies (EBs) and treated with 0.1 mmol/L ascorbic acid to induce the differentiation of ESCs into CMs. In the indirect co-culture system, EBs were co-cultured with epidermal keratinocytes (EKs) or neonatal CMs (NCMs) by the hanging cell culture inserts (PET membranes with 1 µm pores). The molecular expressions and functional properties of ESC-derived CMs in prolonged culture course were evaluated. During time course of ESC differentiation, the percentages of EBs with contracting areas in NCMs co-culture were significantly higher than that without co-culture or in EKs co-culture. The functional maintenance of ESC-derived CMs were more prominent in NCMs co-culture model. CONCLUSIONS/SIGNIFICANCE: These results indicate that NCMs co-culture promote ESC differentiation and has a further effect on cell growth and differentiation. We assume that the improvement of the differentiating efficiency of ESCs into CMs in the co-culture system do not result from the effect of co-culture directly on cell differentiation, but rather by signaling effects that influence the cells in proliferation and long-term function maintenance.

Collagen/ß(1) integrin interaction is required for embryoid body formation during cardiogenesis from murine induced pluripotent stem cells.

BACKGROUND: The interactions between stem cells and extracellular matrix (ECM) mediated by integrins play important roles in the processes that determine stem cell fate. However, the role of ECM/integrin interaction in the formation of embryoid bodies (EBs) during cardiogenesis from murine induced pluripotent stem cells (miPSCs) remains unclear. RESULTS: In the present study, collagen type I and ß(1) integrin were expressed and upregulated synergistically during the formation of miPSC-derived EBs, with a peak expression at day 3 of differentiation. The blockage of collagen/ß(1) integrin interaction by ß(1) integrin blocking antibody resulted in the production of defective EBs that were characterized by decreased size and the absence of a shell-like layer composed of primitive endoderm cells. The quantification of spontaneous beating activity, cardiac-specific gene expression and cardiac troponin T (cTnT) immunostaining showed that the cardiac differentiation of these defective miPSC-derived EBs was lower than that of control EBs. CONCLUSIONS: These findings indicate that collagen/ß(1) integrin interaction is required for the growth and cardiac differentiation of miPSC-derived EBs and will be helpful in future engineering of the matrix microenvironment within EBs to efficiently direct the cardiac fate of pluripotent stem cells to promote cardiovascular regeneration.

High-mobility group box 1 induces calcineurin-mediated cell hypertrophy in neonatal rat ventricular myocytes.

Cardiac hypertrophy is an independent predictor of cardiovascular morbidity and mortality. In recent years, evidences suggest that high-mobility group box 1 (HMGB1) protein, an inflammatory cytokine, participates in cardiac remodeling; however, the involvement of HMGB1 in the pathogenesis of cardiac hypertrophy remains unknown. The aim of this study was to investigate whether HMGB1 is sufficient to induce cardiomyocyte hypertrophy and to identify the possible mechanisms underlying the hypertrophic response. Cardiomyocytes isolated from 1-day-old Sprague-Dawley rats were treated with recombinant HMGB1, at concentrations ranging from 50 ng/mL to 200 ng/mL. After 24 hours, cardiomyocytes were processed for the evaluation of atrial natriuretic peptide (ANP) and calcineurin A expression. Western blot and real-time RT-PCR was used to detect protein and mRNA expression levels, respectively. The activity of calcineurin was also evaluated using a biochemical enzyme assay. HMGB1 induced cardiomyocyte hypertrophy, characterized by enhanced expression of ANP, and increased protein synthesis. Meanwhile, increased calcineurin activity and calcineurin A protein expression were observed in cardiomyocytes preconditioned with HMGB1. Furthermore, cyclosporin A pretreatment partially inhibited the HMGB1-induced cardiomyocyte hypertrophy. Our findings suggest that HMGB1 leads to cardiac hypertrophy, at least in part through activating calcineurin.

Serum omentin-1 levels are inversely associated with the presence and severity of coronary artery disease in patients with metabolic syndrome.

CONTEXT: Omentin-1, an adipokine secreted from visceral adipose tissue, has been reported to be associated with coronary artery disease (CAD) and metabolic disorders. OBJECTIVE: To clarify the relationship between serum omentin-1 levels and the presence and severity of CAD in patients with metabolic syndrome (MetS). METHODS: We measured serum omentin-1 levels in 175 consecutive patients with MetS and in 46 controls. RESULTS: Serum omentin-1 levels are inversely associated with the presence and angiographic severity of CAD in MetS patients. CONCLUSIONS: Serum omentin-1 might be a potential biomarker to predict the development and progression of CAD in MetS patients.

Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes.

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 µm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells.

[The relationship between reactive oxygen species-dependent activation of p38 MAPK and the expression of tumor necrosis factor-α in cultured cardiomyocytes].

AIM: To investigate the role of p38 mitogen-activated protein kinase(MAPK) in lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) expression in neonatal rat cardiomyocytes and to determine the relationship between reactive oxygen species (ROS) and p38 MAPK activation. METHODS: Cardiomyocytes were isolated from neonatal Sprague-Dawley rats and cultured by differential adhesion. Expression of TNF-α was determined in culture medium by ELISA. Activation of p38 MAPK was determined by Western blot analysis with phospho-specific antibody. ROS generation in cardiomyocytes was determined by peroxide specific probe 2', 7'-dichlorofluorescin diacetate (DCF-DA). RESULTS: In cardiomyocytes stimulated with LPS, the content of TNF-α in culture medium correlated with the activity of p38 MAPK in a time-dependent manner. The activation of p38 was observed after stimulation of 1 mg/L LPS for 1 h. TNF-α accumulated significantly in culture medium at 3 h after stimulation of LPS (P<0.05), which was remarkably attenuated by pretreatment with p38 MAPK specific inhibitor SB203580 (P<0.01). Furthermore, the production of ROS in cardiomyocytes stimulated with LPS was also increased at 1 h after stimulation of LPS, consistent with p38 MAPK activity. Pretreatment with antioxidants such as N-acetylcysteine and diphenyleneiodonium significantly inhibited the activation of p38 MAPK compared with LPS control (P<0.05). There was no significance in the activity of p38 MAPK among antioxidants pretreatment and non-LPS control groups. CONCLUSION: The activation of p38 MAPK plays an important role in TNF-α expression in LPS-stimulated cardiomyocytes and the increase of ROS production is prerequisite for the activation of p38 MAPK.

Simvastatin inhibits angiotensin II-induced cardiac cell hypertrophy: role of Homer 1a.

1. The scaffolding protein Homer 1a is constitutively expressed in the myocardium, although its function in cardiomyocytes remains poorly understood. The aim of the present study was to investigate Homer 1a expression in hypertrophic cardiac cells and its role in angiotensin (Ang) II-induced cardiac hypertrophy. 2. After serum starvation for 24 h, cells were treated with 1 micromol/L simvastatin, 100 nmol/L angiotensin (Ang) II or their combination added to Dulbecco's modified Eagle's medium containing 0.5% serum. For combination treatment with AngII plus simvastatin, cells were exposed to simvastatin 12 h before the addition of AngII to the medium and cells were then incubated in the presence of both drugs for a further 24 h. Western blotting was used to determine Homer 1a protein expression. Hypertrophy was evaluated by determining the protein content per cell. 3. Homer 1a protein levels were upregulated following AngII-induced hypertrophy in H9C2 cells and neonatal rat cardiomyocytes, and these increases were augmented by simvastatin pretreatment. Concomitantly, simvastatin pretreatment inhibited extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and AngII-induced hypertrophy. 4. The inhibitory effects of simvastatin against AngII-induced hypertrophy were attenuated by Homer 1a silencing, suggesting that simvastatin suppresses cardiac hypertrophy in a Homer 1a-dependent manner. Furthermore, AngII-induced hypertrophy and ERK1/2 phosphorylation in neonatal rat cardiomyocytes were significantly inhibited following the overexpression of Homer 1a using an adenovirus. 5. These results suggest a possible role for Homer 1a in inhibiting cardiac hypertrophy perhaps in part through inhibition of ERK1/2 activation.

Controlling the in vitro differentiation of embryonic stem cells for myocardial tissue engineering applications.

We studied the differentiation of embryonic stem cells (ESCs) and developed a novel protocol for generating functional cardiomyocytes (CMs) from ESCs by co-culturing these with live cardiac cells. We then evaluated the structural and functional properties of these ESC-derived CMs (ESCMs). An acellular matrix obtained from rabbit heart tissues was used as a scaffold. Then ESCMs were seeded onto the acellular matrix for preliminary tissue engineering applications. We found that by mimicking the cardiac microenvironment, the percentage of beating embryoid bodies (EBs) was much higher and the homogeneity of EBs were significantly improved over that seen in the control group (p<0.001). ESCMs in EBs acquired almost the same structural and functional properties as typical CMs. After implantation, the cells in the EBs rapidly grew and expanded in the extracellular matrix. These results indicate that the differentiation of ESCs can be controlled in a cardiac mimicking microenvironment and that ESCs can be used as an ideal cell source for large-scale tissue engineering applications for the procurement of cardiac muscle.