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.

Docosahexaenoic acid induces glial cell-line derived neurotrophic factor release in C6 glioma cells: Implications of antidepressant effects for docosahexaenoic acid.

Dietary deficiency of n-3 polyunsaturated fatty acids (PUFAs) is involved in the pathophysiology and etiology of major depressive disorder. Supplementation with docosahexaenoic acid (DHA) exerts antidepressant-like effect; however, the molecular mechanism of DHA action remains unclear. Here we examined the effects of DHA on the modulation of glial cell line-derived neurotrophic factor (GDNF), which is essential for neural development, plasticity, neurogenesis, and survival. We demonstrated that DHA treatment significantly increased GDNF release in a concentration dependent manner in rat C6 glioma cells (C6 cells) and primary cultured rat astrocytes, which is also associated with increased expression of GDNF mRNA. Furthermore, the DHA-induced GDNF production was inhibited by mitogen activated protein kinase (MEK) inhibitor and protein kinase C (PKC) inhibitor, but not protein kinase A (PKA) inhibitor and p38 mitogen-activated protein kinase (MAPK) inhibitor. DHA-induced extracellular signal-regulated kinase (ERK) activation is dependent on the PKC, as demonstrated by its reversibility after pretreatment with PKC inhibitor. Moreover, fibroblast growth factor receptor (FGFR inhibitor) but not epidermal growth factor receptor (EGFR) inhibitor blocked the activation of ERK induced by DHA treatment. DHA-induced GDNF production was also blocked by FGFR inhibitor, suggesting that FGFR is also involved in ERK activation-mediated GDNF production induced by DHA. Our study demonstrates that DHA-induced release of GDNF, mediated by PKC and FGFR-dependent on ERK activation, may contribute to the antidepressant-like effect of DHA.

Puerarin Suppresses the Self-Renewal of Murine Embryonic Stem Cells by Inhibition of REST-MiR-21 Regulatory Pathway.

BACKGROUND/AIMS: Puerarin shows a wide range of biological activities, including affecting the cardiac differentiation from murine embryonic stem (mES) cells. However, little is known about its effect and mechanism of action on the self-renewal of mES cells. This study aimed to determine the effect of puerarin on the self-renewal and pluripotency of mES cells and its underlying mechanisms. METHODS: RT-PCR and real-time PCR were used to detect the transcripts of core transcription factors, specific markers for multiple lineages, REST and microRNA-21 (miR-21). Colony-forming assay was performed to estimate the self-renewal capacity of mES cells. Western blotting and wortmannin were employed to explore the role of PI3K/Akt signaling pathway in the inhibitory action of puerarin on REST transcript. Transfected mES cells with antagomir21 were used to confirm the role of miR-21 in the action of puerarin on cell self-renewal. RESULTS: Puerarin significantly decreased the percentage of the self-renewal colonies, and suppressed the transcripts of Oct4, Nanog, Sox2, c-Myc and REST. Besides, PECAM, NCAM and miR-21 were up-regulated both under the self-renewal conditions and at day 4 of differentiation. The PI3K inhibitor wortmannin successfully reversed the mRNA expression changes of REST, Nanog and Sox2. Transfection of antagomir21 efficiently reversed the effects of puerarin on mES cells self-renewal. CONCLUSION: Inhibition of REST-miR-21 regulatory pathway may be the key mechanism of puerarin-induced suppression of mES cells self-renewal.

Traditional Chinese medicine baicalin suppresses mESCs proliferation through inhibition of miR-294 expression.

BACKGROUND: Traditional Chinese herbal medicines (TCMs) have been widely used against a broad spectrum of biological activities, including influencing the cardiac differentiation from mouse embryonic stem cells (mESCs). However, their effects and mechanisms of action on ESCs proliferation remain to be determined. The present study aimed to determine the effect of three TCMs, baicalin, ginsenoside Rg1, and puerarin, on mESCs proliferation and to elucidate the possible mechanism of their action. METHODS: Cell proliferation was examined with a cell proliferation assay Cell Counting Kit-8 (CCK-8), propidium iodide (PI) staining was used to visualize cell cycle. The mRNA expression level of c-myc, c-fos, c-jun, GAPDH and microRNAs were measured by quantitative real time RT-PCR. RESULTS: We found that baicalin 50 µM suppressed the proliferation of mESCs as observations in more cells in G1 phase and less cells in either S phase or G2/M phase. Moreover, baicalin suppressed the expressions of c-jun and c-fos in mESCs and down-regulated the expression of miR-294. Overexpression of miR-294 in mESCs significantly reversed the effects of baicalin both on mESC proliferation and c-fos/c-jun expression. CONCLUSIONS: Baicalin down-regulation of miR-294 may be its key mechanism of action in decreasing mESCs proliferation.

Puerarin facilitates T-tubule development of murine embryonic stem cell-derived cardiomyocytes.

AIMS: The embryonic stem cell-derived cardiomyocytes (ES-CM) is one of the promising cell sources for repopulation of damaged myocardium. However, ES-CMs present immature structure, which impairs their integration with host tissue and functional regeneration. This study used murine ES-CMs as an in vitro model of cardiomyogenesis to elucidate the effect of puerarin, the main compound found in the traditional Chinese medicine the herb Radix puerariae, on t-tubule development of murine ES-CMs. METHODS: Electron microscope was employed to examine the ultrastructure. The investigation of transverse-tubules (t-tubules) was performed by Di-8-ANEPPS staining. Quantitative real-time PCR was utilized to study the transcript level of genes related to t-tubule development. RESULTS: We found that long-term application of puerarin throughout cardiac differentiation improved myofibril array and sarcomeres formation, and significantly facilitated t-tubules development of ES-CMs. The transcript levels of caveolin-3, amphiphysin-2 and junctophinlin-2, which are crucial for the formation and development of t-tubules, were significantly upregulated by puerarin treatment. Furthermore, puerarin repressed the expression of miR-22, which targets to caveolin-3. CONCLUSION: Our data showed that puerarin facilitates t-tubule development of murine ES-CMs. This might be related to the repression of miR-22 by puerarin and upregulation of Cav3, Bin1 and JP2 transcripts.

Effects of puerarin on cardiac differentiation and ventricular specialization of murine embryonic stem cells.

AIMS: It is important to screen and identify chemical compounds to improve the efficiency of cardiac differentiation and specialization of embryonic stem (ES) cells. The objective of this study was to investigate the effect of puerarin, a natural phytoestrogen, on the in vitro cardiac differentiation and ventricular specialization of murine ES cells. METHODS: Cardiac differentiation of murine ES cells was performed by embryoid body (EB)-based differentiation method. Quantitative RT-PCR, flow cytometry and immunofluorescence were employed to identify cardiomyocytes (CMs) derived from murine ES cells (mES-CMs). Patch clamp was used to study the electrophysiological properties of CMs. RESULTS: We found that continuous puerarin treatment significantly increased the population of ES-CMs which express typical cardiac markers and are electrophysiological intact. Puerarin treatment shifted the cardiac phenotype from pacemaker-like cells to ventricular-like cells, which were Mlc2v-positive and present typical ventricular-like AP. Puerarin up-regulated transcripts involved in cardiac differentiation and ventricular specialization of ES cells. CONCLUSION: Our results suggest that puerarin promotes cardiac differentiation, and significantly enhances the specialization of mES cells into ventricular-like CMs. Puerarin may be used to increase the yield of ventricular mES-CMs during in vitro differentiation.

Baicalin maintains late-stage functional cardiomyocytes in embryoid bodies derived from murine embryonic stem cells.

BACKGROUND/AIMS: Low efficiency of cardiomyocyte (CM) differentiation from embryonic stem (ES) cells limits their therapeutic use. The objective of this study was to investigate the effect of baicalin, a natural flavonoid compound, on the in vitro cardiac differentiation of murine ES cells. METHODS: The induction of ES cells into cardiac-like cells was performed by embryoid body (EB)-based differentiation method. The electrophysiological properties of the ES cell-derived CMs (ES-CMs) were measured by patch-clamp. The biomarkers of ES-CMs were determined by quantitative RT-PCR and immunofluorescence. RESULTS: Continuous baicalin treatment decreased the size of EBs, and increased the proportion of α-actinin-positive CMs and transcript level of cardiac specific markers in beating EBs by inducing cell death of non-CMs. Baicalin increased the percentage of working ES-CMs which had typical responses to ß-adrenergic and muscarinic stimulations. CONCLUSION: Baicalin maintains the late-stage functional CMs in EBs derived from murine ES cells. This study describes a new insight into the various biological effects of baicalin on cardiac differentiation of pluripotent stem cells.

Properties and functions of KATP during mouse perinatal development.

BACKGROUND: Prevailing data suggest that ATP-sensitive potassium channels (K(ATP)) contribute to a surprising resistance to hypoxia in mammalian embryos, thus we aimed to characterize the developmental changes of K(ATP) channels in murine fetal ventricular cardiomyocytes. METHODS: Patch clamp was applied to investigate the functions of K(ATP). RT-PCR, Western blot were used to further characterize the molecular properties of K(ATP) channels. RESULTS: Similar K(ATP) current density was detected in ventricular cardiomyocytes of late development stage (LDS) and early development stage (EDS). Molecular-biological study revealed the upregulation of Kir6.1/SUR2A in membrane and Kir6.2 remained constant during development. Kir6.1, Kir6.2, and SUR1 were detectable in the mitochondria without marked difference between EDS and LDS. Acute hypoxia-ischemia led to cessation of APs in 62.5% of tested EDS cells and no APs cessation was observed in LDS cells. SarcK(ATP) blocker glibenclamide rescued 47% of EDS cells but converted 42.8% of LDS cells to APs cessations under hypoxia-ischemic condition. MitoK(ATP) blocker 5-HD did not significantly influence the response to acute hypoxia-ischemia at either EDS or LDS. In summary, sarcK(ATP) played distinct functional roles under acute hypoxia-ischemic condition in EDS and LDS fetal ventricular cardiomyocytes, with developmental changes in sarcK(ATP) subunits. MitoK(ATP) were not significantly involved in the response of fetal cardiomyocytes to acute hypoxia-ischemia and no developmental changes of K(ATP) subunits were found in mitochondria.

[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].

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.

Molecular and functional changes in voltage-gated Na⁺ channels in cardiomyocytes during mouse embryogenesis.

BACKGROUND: Embryonic cardiomyocytes undergo profound changes in their electrophysiological properties during development. However, the molecular and functional changes in Na⁺ channel during cardiogenesis are not yet fully explained. METHODS AND RESULTS: To study the functional changes in the Na⁺ channel during cardiogenesis, Na⁺ currents were recorded in the early (EDS) and late (LDS) developmental stages of cardiomyocytes in embryonic mice. Compared with EDS myocytes, LDS myocytes exhibited a larger peak current density, a more negative shift in the voltage of half inactivation, a larger fast inactivation component and a smaller slow inactivation component, and smaller time constants for recovery from inactivation. Additionally, multiple Na⁺ channel α-subunits (Nav 1.1-1.6) and ß-subunits (Nav ß1-ß3) of mouse embryos were investigated. Transcripts of Nav 1.1-1.3 were absent or present at very low levels in embryonic hearts. Transcripts encoding Nav 1.4-1.6 and Nav ß1-ß3 increased during embryogenesis. Data on the sensitivity of total Na⁺ currents to tetrodotoxin (TTX) showed that TTX-resistant Nav 1.5 is the predominant isoform expressed in the heart of the mouse embryo. CONCLUSIONS: The results indicate that significant changes in the functional properties of Na⁺ channels develop in the cardiomyocytes of the mouse embryo, and that different Na⁺ channel subunit genes are strongly regulated during embryogenesis, which further support a physiological role for voltage-gated Na⁺ channels during heart development.

Functional characterization of inward rectifier potassium ion channel in murine fetal ventricular cardiomyocytes.

AIMS: Previous studies have shown the dramatic changes in electrical properties of murine fetal cardiomyocytes, while details on inward rectifier potassium current (IK1) are still seldom discussed. Thus we aimed to characterize the functional expression and functional role of IK1 in murine fetal ventricular cardiomyocytes. METHODS: Whole cell patch clamp was applied to investigate the electrophysiological properties of IK1. Quantitative real-time PCR, western blotting and double-label immunofluorescence were further utilized to find out the molecular basis of IK1. RESULTS: Compared to early developmental stage (EDS), IK1 at late developmental stage (LDS) displayed higher current density, stronger rectifier property and faster activation kinetics. It was paralleled with the downregulation of Kir2.3 and the upregulation of Kir2.1/Kir2.2. IK1 contributed to maintain the maximum diastolic potential (MDP), late repolarization phase (LRP) as well as the action potential duration (APD). However, the contribution to MDP and velocity of LRP did not change significantly with maturation. CONCLUSIONS: During fetal development, the switch of IK1 subtypes from Kir2.1/Kir2.3 to Kir2.1 resulted in the dramatic changes in IK1 electrophysiological properties.

Hyposmotic challenge modulates function of L-type calcium channel in rat ventricular myocytes through protein kinase C.

AIM: To study the effects and mechanisms by which hyposmotic challenge modulate function of L-type calcium current (I(Ca,L)) in rat ventricular myocytes. METHODS: The whole-cell patch-clamp techniques were used to record I(Ca,L) in rat ventricular myocytes. RESULTS: Hyposmotic challenge(∼220 mosmol/L) induced biphasic changes of I(Ca,L), a transient increase followed by a sustained decrease. I(Ca,L) increased by 19.1%±6.1% after short exposure (within 3 min) to hyposmotic solution. On the contrary, long hyposmotic challenge (10 min) decreased I(Ca,L) to 78.1%±11.0% of control, caused the inactivation of I(Ca,L), and shifted the steady-state inactivation curve of I(Ca,L) to the right. The decreased I(Ca,L) induced by hyposmotic swelling was reversed by isoproterenol or protein kinase A (PKA) activator foskolin. Hyposmotic swelling also reduced the stimulated I(Ca,L) by isoproterenol or foskolin. PKA inhibitor H-89 abolished swelling-induced transient increase of I(Ca,L), but did not affect the swelling-induced sustained decrease of I(Ca,L). NO donor SNAP and protein kinase G (PKG) inhibitor Rp-8-Br-PET-cGMPS did not interfere with swelling-induced biphasic changes of I(Ca,L). Protein kinase C (PKC) activator PMA decreased I(Ca,L) and hyposmotic solution with PMA reverted the decreased I(Ca,L) by PMA. PKC inhibitor BIM prevented the swelling-induced biphasic changes of I(Ca,L). CONCLUSION: Hyposmotic challenge induced biphasic changes of I(Ca,L), a transient increase followed by a sustained decrease, in rat ventricular myocytes through PKC pathway, but not PKG pathway. PKA system could be responsible for the transient increase of I(Ca,L) during short exposure to hyposmotic solution.

Role of transient receptor potential vanilloid 4 in the effect of osmotic pressure on myocardial contractility in rat.

The aim of the present study was to investigate the influence of osmotic pressure on myocardial contractility and the possible mechanism. Electrical stimulation was used to excite papillary muscles of the left ventricle of Sprague-Dawley (SD) rats. The contractilities of myocardium in hyposmotic, isosmotic, and hyperosmotic perfusates were recorded. The influences of agonist and antagonist of the transient receptor potential vanilloid 4 (TRPV4) on the contractility of myocardium under hyposmotic, isosmotic and hyperosmotic conditions were observed. The results were as follows: (1) Compared with that under isosmotic condition (310 mOsm/L), the myocardial contractility was increased by 11.5%, 21.5% and 25.0% (P<0.05) under hyposmotic conditions when the osmotic pressure was at 290, 270 and 230 mOsm/L, respectively; and was decreased by 16.0%, 23.7% and 55.2% (P<0.05) under hyperosmotic conditions when the osmotic pressure was at 350, 370 and 390 mOsm/L, respectively. (2) When ruthenium red (RR), an antagonist of TRPV4, was added to the hyposmotic perfusate (270 mOsm/L), the positive inotropic effect of hyposmia was restrained by 36% (P<0.01); and when RR was added to the hyperosmotic perfusate (390 mOsm/L), the inhibitory effect of hyperosmia on myocardial contractility was increased by 56.1% (P<0.01). (3) When 4-α-phorbol-12,13-didecanoate (4α-PDD), an agonist of TRPV4, was added to the isosmotic perfusate (310 mOsm/L), the myocardial contractility did not change; and when 4α-PDD was added to the hyperosmotic perfusate (390 mOsm/L), the inhibition of myocardial contractility by hyperosmia was increased by 27.1% (P<0.01). These results obtained indicate that TRPV4 is possibly involved in the osmotic pressure-induced inotropic effect.

Cannabinoids inhibit ATP-activated currents in rat trigeminal ganglionic neurons.

The present study aimed to investigate whether cannabinoids could modulate the response mediated by ATP receptor (P2X purinoceptor). Whole-cell patch-clamp recording was performed on cultured rat trigeminal ganglionic (TG) neurons. The majority of TG neurons were sensitive to ATP (67/75, 89.33%). Extracellular pretreatment with WIN55212-2, a cannabinoid receptor 1 (CB1 receptor) agonist, reduced ATP-activated current (I(ATP)) significantly. This inhibitory effect was concentration-dependent and was blocked by AM281, a specific CB1 receptor antagonist. Pretreatment with WIN55212-2 at 1×10(-13), 1×10(-12), 1×10(-11), 1×10(-10), 1×10(-9) and 1×10(-8) mol/L reduced I(ATP) (induced by 1×10(-4) mol/L ATP) by (8.14±3.14)%, (20.11±2.72)%, (46.62±3.51)%, (72.16±5.64)%, (80.21±2.80)% and (80.59±3.55)%, respectively. The concentration-response curves for I(ATP) pretreated with and without WIN55212-2 showed that WIN55212-2 shifted the curve downward, and decreased the maximal amplitude of I(ATP) by (58.02±4.21)%. But the threshold value and EC(50) (1.15×10(-4) mol/L vs 1.27×10(-4) mol/L) remained unchanged. The inhibition of I(ATP) by WIN55212-2 was reversed by AM281, suggesting that the inhibition was mediated via the CB1 receptor. Pretreatment with forskolin [an agonist of adenylyl cyclase (AC)] or 8-Br-cAMP reversed the inhibition of I(ATP) by WIN55212-2. These results suggest that the inhibitory effect of cannabinoids on I(ATP) is mediated via the CB1 receptors, that lead to inhibition of the AC-cAMP-PKA signaling pathway.

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.

Effect of hypoxia on hyperpolarization-activated current in mouse dorsal root ganglion neurons.

The properties of hyperpolarization-activated current (I(h)) in mouse dorsal root ganglion (DRG) neurons and the effect of hypoxia on the current have been studied using whole-cell configuration of the patch clamp technique. Under voltage-clamp mode, I(h), blocked by 1 mM extracellular CsCl, was present in 75.5% of mouse DRG neurons. The distribution rate increased as the neurons become larger, 5.3%, 79.8% and 94.2% in small, medium and large neurons, respectively. Both I(h) density and the rate of I(h) activation increased in response to more hyperpolarized potential. The activation of I(h) current in larger neuron was faster than in smaller neuron, there was a significant correlation between the time constant of I(h) activation and neuron's size. However, I(h) density did not show any correlation with neuron's size. Under current-clamp mode, 'depolarizing sag' was observed in all neurons with I(h) current. The reversal potential (V(rev)) and the maximal conductance density of I(h) (G(h.max-density)) were -31.0 +/- 4.8 mV and 0.17 +/- 0.02 nS/pF, with a half-activated potential (V(0.5) = -99.4 +/- 1.1 mV) and a slope factor (kappa = -10.2 +/- 0.3 mV). There was a correlation between neuron's size and G(h.max-density) only. According to the effect of hypoxia on resting membrane potential, there were hypoxia-sensitive and hypoxia-insensitive neurons. In the hypoxia-sensitive neurons, I(h) was fully abolished by hypoxia, although the resting membrane potential was hyperpolarized. V(0.5) and V(rev) were shifted about 30 mV toward hyperpolarization, whereas G(h.max-density) and kappa were not affected by hypoxia. We suggest that the kinetics and voltage-dependent characteristics of I(h) are varied in mouse DRG neurons with different size. Hypoxia inhibits I(h) in the hypoxia-sensitive neurons by shifting its activation potential to a more hyperpolarized level.

Action of beta-amyloid peptide1₋40 on I(HVA) and its modulation by ginkgolide B.

Whole-cell patch clamp recording was used to investigate the action of beta-amyloid peptide(1-40) (Abeta(1-40)) on high voltage-activated calcium channel current (I(HVA)) in acutely isolated hippocampal CA1 pyramidal neurons in rats and observe its modulation by ginkgolide B (GB). Drug was applied by extracellular bath or adding in the pipette solution, and its effect was determined by comparing the amplitude of I(HVA) before and after the drug application. Bath application of aggregated Abeta(1-40) at concentrations of 0.01~30 mumol/L increased the amplitude of I(HVA) in a dose-dependent manner by (5.43+/-3.01)% (n=8, P>0.05), (10.49+/-4.13) % (n=11, P>0.05), (40.69+/-8.01) % (n=16, P<0.01), (58.32+/-4.85) % (n=12, P<0.01), and (75.45+/-5.81) % (n=6, P<0.01), respectively, but had no effect on the I-V curve of I(HVA); fresh Abeta(1-40) almost had no effect on I(HVA) (n=5, P>0.05). L-type calcium channel antagonist nifedipine abolished the increase of I(HVA)by Abeta(1-40). The increase of I(HVA) by Abeta(1-40) (1.0 mumol/L) was enhanced to (66.19+/-5.74) % (P<0.05) by 8-Br-cAMP (membrane permeable analogue of cAMP) and to (73.21+/-6.90) % (P<0.05) by forskolin, an adenylyl cyclase (AC) agonist, and reduced to (20.08+/-2.18) % (P<0.05) by H-89, cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) antagonist. GB effectively inhibited the increase of I(HVA) by Abeta(1-40). The results indicate that Abeta(1-40) leads to an intracellular calcium overload by increasing I(HVA) via AC-cAMP-PKA. This may be one of the mechanisms for its neurotoxicity. GB can prevent neurons from neurotoxicity by inhibiting abnormal calcium influx caused by Abeta(1-40).

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.

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

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.

Characterization of monoclonal antibody to L-homocysteic acid and its immunohistochemistry in rat hippocampus.

L-homocysteic acid (HCA) and other amino acids were conjugated to rat brain material (extracted rat brain protein) with glutaraldehyde to form HCA- and amino acids-brain material conjugates. The specificity of monoclonal antibody (McAb) was tested on serial dilution test and absorption test on enzyme-linked immunosorbent assay (ELISA) using these conjugates as antigens instead of amino acids-BSA (bovine serum albumin) conjugates used previously. The characterized McAb was applied for immunohistochemical staining using PAP (peroxidase antiperoxidase) technique in combination with silver enhancement of diamino-benzene (DAB) products. The results indicated that McAb to L-HCA reacted with L-HCA-brain material conjugates, but not with other amino acids-brain material conjugates so far tested. McAb absorbed with L-HCA-brain material abolished or decreased immunoreactivity of L-HCA-brain material with McAb. The antibody selectively stained subpopulation of cells and processes in the hippocampus fixed with glutaradehyde. Absorption of McAb with L-HCA-brain material abolished immunohistochemical staining. These results suggested that McAb was specific for L-HCA-brain materials and could be used for imuno-histocytochemistry. This would provide a new tool for immunohistochemical visualization and localization of L-HCA in the nervous system.