Adipose­derived stem cells overexpressing SK4 calcium­activated potassium channel generate biological pacemakers.

Recent studies have suggested that calcium­activated potassium channel (KCa) agonists increase the proportion of mouse embryonic stem cell­derived cardiomyocytes and promote the differentiation of pacemaker cells. In the present study, it was hypothesized that adipose­derived stem cells (ADSCs) can differentiate into pacemaker­like cells via overexpression of the SK4 gene. ADSCs were transduced with a recombinant adenovirus vector carrying the mouse SK4 gene, whereas the control group was transduced with GFP vector. ADSCs transduced with SK4 vector were implanted into the rat left ventricular free wall. Complete atrioventricular block (AVB) was established in isolated perfused rat hearts after 2 weeks. SK4 was successfully and stably expressed in ADSCs following transduction. The mRNA levels of the pluripotent markers Oct­4 and Sox­2 declined and that of the transcription factor Shox2 was upregulated following SK4 transduction. The expression of α­actinin and hyperpolarization­activated cyclic nucleotide­gated potassium channel 4 (HCN4) increased in the SK4 group. The hyperpolarizing activated pacemaker current If (8/20 cells) was detected in ADSCs transduced with SK4, but not in the GFP group. Furthermore, SK4 transduction induced the expression of p­ERK1/2 and p­p38 MAPK. In the ex vivo experiments, the heart rate of the SK4 group following AVB establishment was significantly higher compared with that in the GFP group. Immunofluorescence revealed that the transduced ADSCs were successfully implanted and expressed HCN4 in the SK4 group. In conclusion, SK4 induced ADSCs to differentiate into cardiomyocyte­like and pacemaker­like cells via activation of the extracellular signal­regulated kinase 1/2 and p38 mitogen­activated protein kinase pathways. Therefore, ADSCs transduced with SK4 may be used to generate biological pacemakers in ex vivo rat hearts.

Overexpression of the medium­conductance calcium­activated potassium channel (SK4) and the HCN2 channel to generate a biological pacemaker.

Ion channels serve important roles in the excitation­contraction coupling of cardiac myocytes. Previous studies have shown that the overexpression or activation of intermediate­conductance calcium­activated potassium channel (SK4, encoded by KCNN4) in embryonic stem cell­derived cardiomyocytes can significantly increase their automaticity. The mechanism underlying this effect is hypothesized to be associated with the activation of hyperpolarization­activated cyclic nucleotide­gated channel 2 (HCN2). The aim of the present study was to explore whether a biological pacemaker could be constructed by overexpressing SK4 alone or in combination with HCN2 in a rat model. Ad­green fluorescent protein (GFP), Ad­KCNN4 and Ad­HCN2 recombinant adenoviruses were injected into the left ventricle of Sprague­Dawley rat hearts. The rats were divided into a GFP group (n=10), an SK4 group (n=10), a HCN2 group (n=10) and an SK4 + HCN2 (SK4/HCN2) group (n=10). The isolated hearts were perfused at 5­7 days following injection, and a complete heart block model was established. Compared with the GFP group, overexpressing SK4 alone did not significantly increase the heart rate after establishment of a complete heart block model [98.1±8.9 vs. 96.7±7.6 beats per min (BPM)], The heart rates in the SK4/HCN2 (139.9±21.9 BPM) and HCN2 groups (111.7±5.5 BPM) were significantly increased compared with the GFP and SK4 groups, and the heart rates in the SK4/HCN2 group were significantly increased compared with the SK4 or HCN2 groups. In the HCN2 (n=8) and the SK4/HCN2 (n=7) groups, the shape of the spontaneous ventricular rhythm was the same as the pacing­induced ectopic rhythm in the transgenically altered site. By contrast, these rhythms were different in the SK4 (n=10) and GFP (n=10) groups. There were no significant differences in action potential duration alternans or ventricular arrhythmia inducibility between the four groups (all P>0.05). Western blotting, reverse transcription­quantitative PCR and immunohistochemistry analyses showed that the expression levels of SK4 and HCN2 were significantly increased at the transgene site. Biological pacemaker activity could be successfully generated by co­overexpression of SK4 and HCN2 without increasing the risk of ventricular arrhythmias. The overexpression of SK4 alone is insufficient to generate biological pacemaker activity. The present study provided evidence that SK4 and HCN2 combined could construct an ectopic pacemaker, laying the groundwork for the development of improved biological pacing mechanisms in the future.

Insulin gene enhancer binding protein 1 induces adipose tissue­derived stem cells to differentiate into pacemaker­like cells.

Hybrid approaches combining gene­ and cell­based therapies to make biological pacemakers are a promising therapeutic avenue for bradyarrhythmia. The present study aimed to direct adipose tissue­derived stem cells (ADSCs) to differentiate specifically into cardiac pacemaker cells by overexpressing a single transcription factor, insulin gene enhancer binding protein 1 (ISL­1). In the present study, the ADSCs were transfected with ISL­1 or mCherry fluorescent protein lentiviral vectors and co­cultured with neonatal rat ventricular cardiomyocytes (NRVMs) in vitro for 5­7 days. The feasibility of regulating the differentiation of ADSCs into pacemaker­like cells by overexpressing ISL­1 was evaluated by observation of cell morphology and beating rate, reverse transcription­quantitative polymerase chain reaction analysis, western blotting, immunofluorescence and analysis of electrophysiological activity. In conclusion, these data indicated that the overexpression of ISL­1 in ADSCs may enhance the pacemaker phenotype and automaticity in vitro, features which were significantly increased following co­culture induction.

3,3'-Diindolylmethane protects cardiomyocytes from LPS-induced inflammatory response and apoptosis.

BACKGROUND: 3,3'-Diindolylmethane (DIM) has been extensively studied as a potential therapeutic drug with free radical scavenging, antioxidant and anti-angiogenic effects. However, whether DIM has similar effects on cardiomyocytes remains unknown. Here we evaluated DIM's influence on inflammation and apoptosis of H9C2 cardiomyocytes induced by LPS and to explore the possible mechanism of the effects. METHODS: H9C2 cells were incubated with DIM (10, 20 and 30 µM) with or without LPS for 24 h. The cytotoxicity of DIM was detected by CCK-8. The levels of tumour necrosis factor (TNF)-α and interleukin (IL)-6 were then measured using RT-qPCR and ELISA. Cell apoptosis rate and reactive oxygen species (ROS) content after DIM treatment were measured by flow cytometry. Expressions of NFκB, P-NFκB, IκBa, P-IκBa, Bax and Bcl-2 after DIM treatment were detected by western blot. The rate of NFκB nuclear translocation after DIM treatment was determined by immunocytochemical analysis. RESULTS: LPS stimulation promoted TNF-α and IL-6 mRNA expression. After treatment with various concentrations of DIM (10, 20 and 30 µM), TNF-α and IL-6 mRNA expression was clearly impaired, especially in the LPS + DIM30(µM) group. ELISA was used to measure TNF-α and IL-6 concentrations in cellular supernatant, and the result was verified to be consistent with RT-qPCR. Additionally, DIM treatment significantly blocked LPS-induced oxidative stress and inhibited LPS-induced apoptosis in H9C2 cardiomyocytes according to the results detected by flow cytometry. Moreover, compared with LPS alone, DIM significantly inhibited the LPS-induced phosphorylation of NFκB (p-NFκB) and Bax expression and increased Bcl-2 expression. CONCLUSIONS: DIM may have a protective effect for H9C2 cardiomyocytes against LPS-induced inflammatory response and apoptosis. DIM may be a new insight into the treatment of septic cardiomyopathy.

Sensitive electrochemical immunosensor for α-fetoprotein based on graphene/SnO2/Au nanocomposite.

A label-free electrochemical immunosensor for sensitive detection of α-fetoprotein (AFP) was developed based on graphene/SnO2/Au nanocomposite. The graphene/SnO2/Au nanocomposite modified glassy carbon electrode was used to immobilize α-fetoprotein antibody (anti-AFP) and to construct the immunosensor. Results demonstrated that the peak currents of [Ru(NH3)6](3+) decreased due to the interaction between antibody and antigen on the modified electrode. Thus, a label-free immunosensor for the detection of AFP was realized by monitoring the peak current change of [Ru(NH3)6](3+). The factors influencing the performance of the immunosensor were investigated in details. Under optimal conditions, the peak currents obtained by DPV decreased linearly with the increasing AFP concentrations in the range from 0.02 to 50 ng mL(-1) with a linear coefficient of 0.9959. This electrochemical immunoassay has a low detection limit of 0.01 ng mL(-1) (S/N=3) and was successfully applied to the determination of AFP in serum samples.

A simple one-pot synthesis of graphene nanosheet/SnO2 nanoparticle hybrid nanocomposites and their application for selective and sensitive electrochemical detection of dopamine.

A novel one-pot synthesis of graphene nanosheet/SnO2 nanoparticle hybrid nanocomposites (GN/SnO2) was realized by using graphene oxide nanosheets (GONs) functionalized with sodium dodecyl sulfonate and SnCl2 as the starting materials. The morphology and structure of the synthesized SDS-GN/SnO2 nanocomposites were characterized by Raman spectroscopy, transmission electron microscopy (TEM) and X-ray diffraction analysis. It was found that SnO2 nanoparticles were homogeneously distributed on the graphene nanosheets. The electrochemical behavior of dopamine (DA) at the SDS-GN/SnO2 nanoparticle modified electrode was studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The results showed that the modified electrode exhibited excellent electrocatalytic activity towards the electrochemical oxidation of DA. The separation of the oxidation peak potentials for ascorbic acid (AA)-DA, uric acid (UA)-DA and UA-AA obtained by DPV is about 132 mV, 128 mV and 260 mV, respectively, which allows selective and sensitive detection of DA in the presence of AA and UA. The anodic peak currents were linear with the concentration of DA in the range from 1.0 × 10-7 to 1.0 × 10-5 M with a coefficient of 0.9980. The detection limit was 80 nM (S/N = 3). The proposed method could be applied for the determination of DA in real human urine samples.