IL-6 promotes pluripotency of mouse embryonic stem cells and regulates cardiac differentiation in a development-dependent manner / 生理学报

Interleukin 6 (IL-6), an important component of cardiac microenvironment, favors cardiac repair by improving cardiomyocyte regeneration in different models. This study aimed to investigate the effects of IL-6 on stemness maintenances and cardiac differentiation of mouse embryonic stem cells (mESCs). The mESCs were treated with IL-6 for two days, and then subjected to CCK-8 essay for proliferation analysis and quantitative real-time PCR (qPCR) to evaluate the mRNA expression of genes related to stemness and germinal layers differentiation. Phosphorylation levels of stem cell-related signal pathways were detected by Western blot. siRNA was used to interfere the function of STAT3 phosphorylation. Cardiac differentiation was investigated by the percentage of beating embryoid bodies (EBs) and qPCR analysis of cardiac progenitor markers and cardiac ion channels. IL-6 neutralization antibody was applied to block the endogenous IL-6 effects since the onset of cardiac differentiation (embryonic day of 0, EB0). The EBs were collected on EB7, EB10 and EB15 to investigate the cardiac differentiation by qPCR. On EB15, Western blot was applied to investigate the phosphorylation of several signaling pathways, and immunochemistry staining was adopted to trace the cardiomyocytes. IL-6 antibody was administered for two days (short term) on EB4, EB7, EB10 or EB15, and percentages of beating EBs at late developmental stage were recorded. The results showed that exogenous IL-6 promoted mESCs proliferation and favored maintenances of pluripotency, evidenced by up-regulated mRNA expression of oncogenes (c-fos, c-jun) and stemness markers (oct4, nanog), down-regulated mRNA expression of germ layer genes (branchyury, FLK-1, pecam, ncam, sox17), and increased phosphorylation of ERK1/2 and STAT3. siRNA targeting JAK/STAT3 partially attenuated the effects of IL-6 on cell proliferation and mRNA expression of c-fos and c-jun. During differentiation, long term IL-6 neutralization antibody application decreased the percentage of beating EBs, down-regulated mRNA expression of ISL1, GATA4, α-MHC, cTnT, kir2.1, cav1.2, and declined the fluorescence intensity of cardiac α actinin in EBs and single cell. Long term IL-6 antibody treatment decreased the phosphorylation of STAT3. In addition, short term (2 d) IL-6 antibody treatment starting from EB4 significantly reduced the percentage of beating EBs in late development stage, while short term IL-6 antibody treatment starting from EB10 significantly increased the percentage of beating EBs on EB16. These results suggest that exogenous IL-6 promotes mESCs proliferation and favors stemness maintenance. Endogenous IL-6 regulates mESC cardiac differentiation in a development-dependent manner. These findings provide important basis for the study of microenvironment on cell replacement therapy, as well as a new perspective for understanding the pathophysiology of heart diseases.

Cardiac fibroblast paracrine factors modulate mouse embryonic stem cells / 生理学报

The study aims to investigate the effects of cardiac fibroblast (CF) paracrine factors on murine embryonic stem cells (ESCs). Conditioned mediums from either neonatal cardiac fibroblasts (ConM-NCF) or adult cardiac fibroblasts (ConM-ACF) were diluted by 1:50 and 1:5, respectively, to investigate whether these conditioned mediums impact murine ESCs distinctly with RT-real time PCR techniques, cell proliferation essay, ELISA and by counting percentage of beating embryoid bodies (EBs) during ESCs differentiation. The data showed that the paracrine ability of CFs changed dramatically during development, in which interleukin 6 (IL6) increased with maturation. ConM-NCF 1:50 and ConM-NCF 1:5 had opposite effects on the pluripotent markers, although they both reduced mouse ESC proliferation. ConM-ACF 1:50 promoted ESCs pluripotent markers and proliferation, while ConM-ACF 1:5 exerted negative effects. All CF-derived conditioned mediums inhibited cardiac differentiation, but with distinguishable features: ConM-NCF 1:50 slightly decreased the early cardiac differentiation without altering the maturation tendency or cardiac specific markers in EBs at differentiation of day 17; ConM-ACF 1:50 had more significant inhibitory effects on early cardiac differentiation than ConM-NCF 1:50 and impeded cardiac maturation with upregulation of cardiac specific markers. In addition, IL6 neutralization antibody attenuated positive effect of ConM-ACF 1:50 on ESCs proliferation, but had no effects on ConM-NCF 1:50. Long-term IL6 neutralization reduced the percentage of beating EBs at early developmental stage, but did not alter the late cardiac differentiation. Taken together, both the quality and quantity of factors and cytokines secreted by CFs are critical for the ESC fate. IL6 could be a favorable cytokine for ESC pluripotency and the early cardiac differentiation.

Thymosin β4 impeded murine stem cell proliferation with an intact cardiovascular differentiation / 华中科技大学学报（医学）（英德文版）

Thymosin β4 (Tβ4) 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 Tβ4 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 Tβ4 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 Tβ4 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 Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 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 Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.

Expression of Kir2.1, SCN5a and SCN1b channel genes in mouse cardiomyocytes with various electric properties: patch clamp combined with single cell RT-PCR study / 生理学报

This study is to explore a new method of investigating molecular basis for electrophysiological properties of early fetal cardiomyocytes. Single embryonic cardiomyocytes of mouse early developmental heart (E10.5) were obtained by a collagenase B digestion approach. After recording spontaneous action potential using whole cell patch clamp technique, the single cell was picked by a glass micropipette, followed by a standard RT-PCR to explore the expression levels of several ion channel genes. Three phenotypes of cardiomyocytes were demonstrated with distinct properties: ventricular-like, atrial-like, and pacemaker-like action potentials. Ventricular-like and atrial-like cells were characterized with much negative maximum diastolic potential (MDP) and a higher V(max) (maximum velocity of depolarization) compared to pacemaker-like cells. MDP of ventricular-like cells was the most negative. In parallel, stronger expression of SCN5a, SCN1b and Kir2.1 were observed in ventricular-like and atrial-like cells compared to that of pacemaker-like cells, where Kir2.1 in ventricular-like cells was the most abundant. Cardiomyocytes with distinct electrophysiological properties had distinct gene expression pattern. Single cell RT-PCR combined with patch clamp technique could serve as a precise detector to analyze the molecular basis of the special electrophysiological characteristics of cardiomyocytes.

Investigation on spontaneous electrical activity of murine embryonic heart using microelectrode arrays / 生理学报

In our studies, we have applied a novel tool, microelectrode arrays (MEA), to investigate the electrophysiological properties of murine embryonic hearts in vitro. The electrical signals were recorded from the areas of the heart adhering to the 60 MEA electrodes, being called field potentials (FPs). As an extracelluar recording, the waveform of the FP appeared similar to a reversed action potential obtained from single cell by whole cell current clamp and the FP duration was comparable with the action potential duration. To study propagation of spontaneous electrical activity, we have compared the occurrence time of FPs recorded from different electrodes. It is shown that there was already an apparent A-V delay [(50.21+/-9.7) ms] at day 9.5 post coitum (E9.5) when heart was still tubular-like and atrium and ventricle were not separated anatomically, while occurence of FP at different electrodes of ventricular area were almost synchronous. Further, we looked into the modulation of spontaneous electrical activity during cardiac development: at E9.5 of embryonic development, 1 mumol/L of isoproterenol (Iso) increased beating frequency by (34.04+/-7.31)%, shortened the A-V delay by (20.00+/-6.44) % and prolonged FP duration. In contrast, 1 mumol/L of carbachol (CCh) slowed down beating frequency by (42.32+/-5.36) %, A-V conduction by (26.00+/-4.81) % and shortened FP duration; however at late stage (E16.5), the regulatory effect of Iso and CCh was strengthened. Therefore we conclude that cardiac conduction system is already established at E9.5 when the four-chambered heart is not formed yet and the regulation of spontaneous activity by sympathetic and para-sympathetic system is gradually matured during cardiac development.

Different signal molecules involved in the muscarinic modulation of pacemaker current I(f) on the heart of mouse embryo in different developmental stages / 生理学报

We isolated mouse embryonic cardiomyocytes derived from timed-pregnant females at different periods and used patch-clamp technique to investigate the muscarinic cholinergic modulation of pacemaker current I(f) in different developmental stages. In early development stage (EDS), muscarinic agonist carbachol (CCh) significantly decreased the magnitude of the pacemaker current I(f) but had no effect in late development stage (LDS). Forskolin (a direct adenylate cyclase activator) and IBMX (a non-selective phosphodiesterase inhibitor) increased I(f) in both EDS and LDS cells. Interestingly, although both forskolin and IBMX increased basal I(f), their effects on CCh-inhibited I(f) were different. Forskolin did not reverse the inhibitory action of CCh until intermediate development stage (IDS). In contrast, IBMX reversed the inhibitory action of CCh on I(f) in EDS but not in IDS. It is suggested that a decrease in intracellular cAMP is a possible mechanism for CCh to modulate I(f). During the EDS and IDS CCh controls the cytoplasmic cAMP level by different pathways: In EDS, CCh modulates I(f) possibly by activating PDE which accelerates the breakdown of cAMP, but in IDS possibly by inhibiting adenylate cyclase (AC) which then reduces the synthesis of cAMP.

Isolation and electrophysiological characteristics of embryonic cardiomyocytes in mice / 生理学报

To explore the electrophysiological characteristics of embryonic cardiomyocytes, single embryonic cardiomyocytes were obtained from mice at different periods by a collegenase B digestion approach, whole cell patch clamp recording technique was used to record I(f) and I(Ca-L), and spontaneous action potential was also recorded. The morphological and spontaneous contractile properties of the isolated cells appeared to be typical embryonic cardiomyocytes when the cells were assessed by phase-contrast microscope. Whole cell recording of isolated cells is easily performed by the whole cell patch clamp technique. Elelctrophysiological properties of I(f) and I(Ca-L) from embryonic cardiomyocytes have been proved to be similar to those from adult pacemaker cells or cardiomyocytes. The established method of isolation is simple, stable, effective and reliable. It allows to obtain as early as 8.5-day embryonic myocytes. The electrophysiological recording of embryonic cardiomyocytes will provide a useful model for exploring the electrophysiological characteristics of embryonic cardiomyocytes and the possible mechanism underlying some heart diseases.