Functional roles of sodium-calcium exchange in autorhythmicity and action potential of murine fetal cardiomyocytes at early developmental stage.

The aim of the present paper was to study the role of sodium calcium exchanger (NCX) in the generation of action potentials (APs) in cardiomyocytes during early developmental stage (EDS). The precisely dated embryonic hearts of C57 mice were dissected and enzymatically dissociated to single cells. The changes of APs were recorded by whole-cell patch-clamp technique before and after administration of NCX specific blockers KB-R7943 (5 µmol/L) and SEA0400 (1 µmol/L). The results showed that, both KB-R7943 and SEA0400 had potent negative chronotropic effects on APs of pacemaker-like cells, while such effects were only observed in some ventricular-like cardiomyocytes. The negative chronotropic effect of KB-R7943 on ventricular-like cardiomyocytes was accompanied by shortening of AP duration (APD), whereas such an effect of SEA0400 was paralleled by decrease in velocity of diastolic depolarization (Vdd). From embryonic day 9.5 (E9.5) to E10.5, the negative chronotropic effects of KB-R7943 and SEA0400 on ventricular-like APs of embryonic cardiomyocytes gradually disappeared. These results suggest that, in the short-term development of early embryo, the function of NCX may experience developmental changes as evidenced by different roles of NCX in autorhythmicity and APs generation, indicating that NCX function varies with different conditions of cardiomyocytes.

MiR-20 regulates myocardiac ischemia by targeting KATP subunit Kir6.1.

This study aimed to examine the functional role of microRNA-20 (miR-20) and its potential target, Kir6.1, in ischemic myocardiocytes. The expression of miR-20 was detected by real-time PCR. Myocardiocytes were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TU-NEL) reagent for apoptosis evaluation. Western blotting was used to detect the Kir6.1 protein in ischemic myocardiocytes transfected with miR-20 mimics or inhibitors. Luciferase reporter gene assay was performed to confirm the targeting effect of miR-20 on KCNJ8. The results showed that miR-20 was remarkably down-regulated, while the KATP subunit Kir6.1 was significantly up-regulated, during myocardial ischemia. The miR-20 overexpression promoted the apoptosis of ischemic myocardiocytes, but showed no such effect on normal cells. Under ischemic condition, myocardiocytes transfected with miR-20 mimics expressed less Kir6.1. On the contrary, inhibiting miR-20 increased the expression of Kir6.1 in the cells. Co-transfection of miR-20 mimics with the KCNJ8 3'-UTR plasmid into HEK293 cells consistently produced less luciferase activity than transfection of the plasmid alone. It was concluded that miR-20 may regulate myocardiac ischemia by targeting KATP subunit Kir6.1 to accelerate the cell apoptosis. Therefore miR-20 may serve as a therapeutic target for myocardial ischemic disease.

Thymosin ß4 impeded murine stem cell proliferation with an intact cardiovascular differentiation.

Thymosin ß4 (Tß4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tß4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tß4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tß4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tß4 with upregulation of Tcf3 and constant ß-catenin. Under mESCs differentiation, Tß4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tß4. In conclusion, Tß4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tß4 did not influence the in vitro cardiovascular differentiation.

A rabbit pulmonary vein myocyte isolation method based on simultaneous heart and pulmonary vein perfusion.

Myocytes in the pulmonary veins (PV) play a pivotal role in the development of paroxysmal atrial fibrillation (AF). It is therefore important to understand physiological characteristics of these cells. Studies on these cells are, however, markedly impeded by the fact that single PV myocytes are very difficult to obtain due to lack of effective isolation methods. In this study, we described a novel PV myocyte isolation method. The key aspect of this method is to establish a combination of retrograde heart perfusion (via the aorta) and anterograde PV perfusion (via the pulmonary artery). With this simultaneous perfusion method, a better perfusion of the PV myocytes can be obtained. As results, the output and viability of single myocytes isolated by simultaneous heart and PV perfusion method were increased compared with those in conventional retrograde heart perfusion method.

[Influence of PAF receptors on long term potentiation attenuated by aluminium in hippocampal CA3 area of rats].

AIM: To investigate the influence of platelet-activating factor (PAF) receptor on long-term potentiation (LTP) attenuated by aluminium. METHODS: The method of extracellular recording was used to investigate the effect of PAF receptors on PP-CA3 LTP by microinjection of PAF receptor antagonist Ginkgolide B or agonist mc-PAF into CA3 area. RESULTS: (1) Amplitude of population spikes (PS) of evoked potential was not affected but LTP induction was blocked by 0.2 micromol/L ginkgolide B in CA3 area. (2) LTP induction was not influenced by 0.25 mol/L aluminium chloride, however, it could be blocked when aluminium was applicated with ginkgolide B. (3) LTP induction was influenced slightly by 40 micromol/L mc-PAF but it has no difference in statistic. LTP induction could be blocked completely by 0.5 mol/L aluminium, but when aluminium was coapplicated with mc-PAF, this effect could be relieved. CONCLUSION: These results indicate that PAF receptors are involved in induction of LTP in CA3 area by stimulating perforant path. The inhibitory effect of aluminium on LTP is partly related to PAF receptors.