Sacubitril/valsartan reduces susceptibility to atrial fibrillation by improving atrial remodeling in spontaneously hypertensive rats.

AIM: Sacubitril/valsartan (Sac/Val, LCZ696), the world's first angiotensin receptor-neprilysin inhibitor (ARNi), has been widely used in the treatment of heart failure. However, the use of Sac/Val in the treatment of atrial fibrillation (AF), especially AF with hypertension, has been less reported. We investigated the effect of Sac/Val on atrial remodeling and hypertension-related AF. METHODS: The AF induction rate and electrophysiological characteristics of spontaneously hypertensive rats (SHRs) treated with Sac/Val or Val were detected by rapid atrial pacing and electrical mapping/optical mapping. The whole-cell patch-clamp and Western blot were used to observe electrical/structural remodeling of atrial myocytes/tissue of rats and atrium-derived HL-1 cells cultured under 40 mmHg in vitro. RESULTS: Sac/Val was superior to Val in reducing blood pressure, myocardial hypertrophy and susceptibility of AF in SHRs. The shorten action potentials duration (APD), decreased L type calcium channel current (ICa,L) and Cav1.2, increased ultrarapid delayed rectified potassium current (Ikur) and Kv1.5 in atrial myocytes/tissue of SHRs could be better improved by Sac/Val, as well as the levels of atrial fibrosis. While the protein expression of angiotensin-converting enzyme-1 (ACE-1), angiotensin, angiotensin II type I AT1 receptor (AT1R) and neprilysin (NEP) were increased, which could be more effective ameliorated by Sac/Val than Val. Furthermore, Val + Sacubitrilat (LBQ657) (an active NEP inhibitor) was also superior to LBQ657 or Val in improving the electrical and structural remodeling of HL-1 cells through inhibiting NEP. CONCLUSION: Sac/Val can improve atrial structural and electrical remodeling induced by hypertension and reduce the AF susceptibility by inhibiting RAS and NEP. The above effects of Sac/Val were superior to Val alone.

Acetyltransferase p300 regulates atrial fibroblast senescence and age-related atrial fibrosis through p53/Smad3 axis.

Atrial fibrosis induced by aging is one of the main causes of atrial fibrillation (AF), but the potential molecular mechanism is not clear. Acetyltransferase p300 participates in the cellular senescence and fibrosis, which might be involved in the age-related atrial fibrosis. Four microarray datasets generated from atrial tissue of AF patients and sinus rhythm (SR) controls were analyzed to find the possible relationship of p300 (EP300) with senescence and fibrosis. And then, biochemical assays and in vivo electrophysiological examination were performed on older AF patients, aging mice, and senescent atrial fibroblasts. The results showed that (1) the left atrial tissues of older AF patients, aging mouse, and senescence human atrial fibroblasts had more severe atrial fibrosis and higher protein expression levels of p300, p53/acetylated p53 (ac-p53)/p21, Smad3/p-Smads, and fibrosis-related factors. (2) p300 inhibitor curcumin and p300 knockdown treated aging mouse and senescence human atrial fibroblasts reduced the senescence ratio of atrial fibroblasts, ameliorated the atrial fibrosis, and decreased the AF inducibility. In contrast, over-expression of p300 can lead to the senescence of atrial fibroblasts and atrial fibrosis. (3) p53 knockdown decreased the expression of aging and fibrosis-related proteins. (4) Co-immunoprecipitation and immunofluorescence showed that p53 forms a complex with smad3 and directly regulates the expression of smad3 in atrial fibroblasts. Our findings suggest that the mechanism of atrial fibrosis induced by aging is, at least, partially dependent on the regulation of p300, which provides new sights into the AF treatment, especially for the elderly.

Advanced glycation end products induce senescence of atrial myocytes and increase susceptibility of atrial fibrillation in diabetic mice.

Diabetes mellitus (DM) is a common chronic metabolic disease caused by significant accumulation of advanced glycation end products (AGEs). Atrial fibrillation (AF) is a common cardiovascular complication of DM. Here, we aim to clarify the role and mechanism of atrial myocyte senescence in the susceptibility of AF in diabetes. Rapid transesophageal atrial pacing was used to monitor the susceptibility of mice to AF. Whole-cell patch-clamp was employed to record the action potential (AP) and ion channels in single HL-1 cell and mouse atrial myocytes. More importantly, anti-RAGE antibody and RAGE-siRNA AAV9 were used to investigate the relationship among diabetes, aging, and AF. The results showed that elevated levels of p16 and retinoblastoma (Rb) protein in the atrium were associated with increased susceptibility to AF in diabetic mice. Mechanistically, AGEs increased p16/Rb protein expression and the number of SA-ß-gal-positive cells, prolonged the action potential duration (APD), reduced protein levels of Cav1.2, Kv1.5, and current density of ICa,L , IKur in HL-1 cells. Anti-RAGE antibody or RAGE-siRNA AAV9 reversed these effects in vitro and in vivo, respectively. Furthermore, downregulating p16 or Rb by siRNA prevented AGEs-mediated reduction of Cav1.2 and Kv1.5 proteins expression. In conclusion, AGEs accelerated atrial electrical remodeling and cellular senescence, contributing to increased AF susceptibility by activating the p16/Rb pathway. Inhibition of RAGE or the p16/Rb pathway may be a potential therapeutic target for AF in diabetes.

Activation of PKCα participates in the reduction of Ikur in atrial myocytes induced by tumour necrosis factor-α.

The atrial-specific ultra-rapid delayed rectifier K+ current (Ikur) plays an important role in the progression of atrial fibrillation (AF). Because inflammation is known to lead to the onset of AF, we aimed to investigate whether tumour necrosis factor-α (TNF-α) played a role in regulating Ikur and the potential signalling pathways involved. Whole-cell patch-clamp and biochemical assays were used to study the regulation and expression of Ikur in myocytes and in tissues from left atrial appendages (LAAs) obtained from patients with sinus rhythm (SR) or AF, as well as in rat cardiomyocytes (H9c2 cells) and mouse atrial myocytes (HL-1 cells). Ikur current density was markedly reduced in atrial myocytes from AF patients compared with SR controls. Reduction of Kv1.5 protein levels was accompanied by increased expression of TNF-α and protein kinase C (PKC)α activation in AF patients. Treatment with TNF-α dose-dependently reduced Ikur and protein expression of Kv1.5 but not Kv3.1b in H9c2 cells and HL-1 cells. TNF-α also increased activity of PKCα. Specific PKCα inhibitor Gö6976 alleviated the reduction in Ikur induced by TNF-α, but not the reduction in Kv1.5 protein. TNF-α was involved in the electrical remodelling associated with AF, probably by depressing Ikur in atrial myocytes via activation of PKCα.

High hydrostatic pressure induces atrial electrical remodeling through angiotensin upregulation mediating FAK/Src pathway activation.

Hypertension is an independent risk factor for atrial fibrillation (AF), although its specific mechanisms remain unclear. Previous research has been focused on cyclic stretch, ignoring the role of high hydrostatic pressure. The present study aimed to explore the effect of high hydrostatic pressure stimulation on electrical remodeling in atrial myocytes and its potential signaling pathways. Experiments were performed on left atrial appendages from patients with chronic AF or sinus rhythm, spontaneously hypertensive rats (SHRs) treated with or without valsartan (10 mg/kg/day) and HL-1 cells were exposed to high hydrostatic pressure using a self-developed device. Whole-cell patch-clamp recordings and western blots demonstrated that the amplitudes of ICa,L, Ito, and IKur were reduced in AF patients with corresponding changes in protein expression. Angiotensin protein levels increased and Ang1-7 decreased, while focal adhesion kinase (FAK) and Src kinase were enhanced in atrial tissue from AF patients and SHRs. After rapid atrial pacing, AF inducibility in SHR was significantly higher, accompanied by a decrease in ICa,L, upregulation of Ito and IKur, and a shortened action potential duration. Angiotensin upregulation and FAK/Src activation in SHR were inhibited by angiotensin type 1 receptor inhibitor valsartan, thus, preventing electrical remodeling and reducing AF susceptibility. These results were verified in HL-1 cells treated with high hydrostatic pressure, and demonstrated that electrical remodeling regulated by the FAK-Src pathway could be modulated by valsartan. The present study indicated that high hydrostatic pressure stimulation increases AF susceptibility by activating the renin-angiotensin system and FAK-Src pathway in atrial myocytes.

High hydrostatic pressure induces atrial electrical remodeling through upregulation of inflammatory cytokines.

AIMS: Hypertension is an independent risk factor for atrial fibrillation (AF). However, the direct effect of hydrostatic pressure on atrial electrical remodeling is unclear. The present study investigated whether hydrostatic pressure is responsible for atrial electrical remodeling and addressed a potential role of inflammation in this pathology. MAIN METHODS: Whole-cell patch-clamp recordings and biochemical assays were used to study the regulation and expression of ion channels in left atrial appendages in patients with AF, spontaneously hypertensive rats (SHRs), and atrium-derived cells (HL-1 cells) exposed to standard (0 mmHg) and elevated (20, 40 mmHg) hydrostatic pressure. KEY FINDINGS: Both TNF-α and MIF were highly expressed in patients with AF and SHRs. AF inducibility in SHRs was higher after atrial burst pacing, accompanied by a decrease in the L-type calcium current (ICa,L), an increase in the transient outward K+ current (Ito) and ultra-rapid delayed rectifier K+ current (IKur), and a shortened action potential duration (APD), which could be inhibited by atorvastatin. Furthermore, exposure to elevated pressure was associated with electrical remodeling of the HL-1 cells. The peak current density of ICa,L was reduced, while Ito and IKur were increased. Moreover, the expression levels of Kv4.3, Kv1.5, TNF-α, and MIF were upregulated, while the expression of Cav1.2 was downregulated in HL-1 cells after treatment with high hydrostatic pressure (40 mmHg). Atorvastatin alleviated the electrical remodeling and increased inflammatory markers in HL-1 cells induced by high hydrostatic pressure. SIGNIFICANCE: Elevated hydrostatic pressure led to atrial electrical remodeling and increased AF susceptibility by upregulating inflammation.

Atorvastatin ameliorates the contractile dysfunction of the aorta induced by organ culture.

Statins are widely used in the treatment of hypercholesterolemia. Studies have demonstrated that statins could maintain vascular contractile function through inhibiting the transformation of vascular smooth muscle cells (VSMCs) from the contractile phenotype to the synthetic phenotype. However, the underlying mechanisms have not been fully elucidated. The effect of atorvastatin on the thoracic aorta of Sprague-Dawley rats cultured in serum-free conditions in vitro was evaluated. Aortic constriction was induced by high potassium, phenylephrine, and CaCl2. The protein expression levels of α1 adrenoceptor; inositol 1,4,5-trisphosphate (IP3) receptor; protein kinase Cδ (PKCδ); stromal interaction molecule 1 (STIM1); high-voltage activated dihydropyridine-sensitive (L type, Cav1.2) channels; and two contractile phenotype marker proteins [α-smooth muscle actin (α-SMA) and myosin (SM-MHC)] were determined by western blotting. Compared with the fresh control, the constriction of rat aorta was impaired after culture in serum-free medium for 24 h. The impaired contraction of cultured aortas was mediated by Cav1.2 and store-operated Ca2+ (SOC) channel, which could be improved by atorvastatin at 20 µM. The protein expression levels of α1 adrenoceptor, IP3 receptor, PKCδ, STIM1, Cav1.2, α-SMA, and SM-MHC in the aortas cultured in serum-free conditions were decreased significantly. Atorvastatin partially prevented the reduction in the contractility and the downregulation of these proteins in cultured aortas. The transformation of the VSMC phenotype is associated with the vasoconstriction dysfunction of cultured aortas. Atorvastatin may protect vascular function by modulating calcium signaling pathways.

[Effects of propofol combined with indomethacin on contraction of isolated human pulmonary arteries].

OBJECTIVE: To investigate the effects of propofol combined with indomethacin on the contractile function of isolated human pulmonary arteries. METHODS: Human pulmonary artery preparations were obtained from patients undergoing surgery for lung carcinoma. The intrapulmonary arteries were dissected and cut into rings under microscope for treatment with propofol or propofol combined with indomethacin. In each group, the rings were divided into endothelium-intact and endothelium-denuded groups and mounted in a Multi Myograph system. In propofol group, the rings were preconstricted by U46619 to induce a sustained contraction, and propofol (10-300 mmol/L) was then applied cumulatively. In the combined treatment group, the rings were pretreated with indomethacin (100 µmol/L) for 30 min before application of U46619 to induce sustained contraction, and propofol (10-300 µmol/L) was added cumulatively after the tension became stable. RESULTS: Propofol (10-100 µmol/L) induced constrictions at low concentrations and caused relaxations at higher concentrations (100-300 µmol/L) in the pulmonary artery rings with prior U46619-induced contraction. Propofol caused stronger constrictions in endothelium-intact rings [EC50=4.525∓0.37, Emax=(30.44∓2.92)%] than in endothelium-denuded rings [EC50=4.699∓0.12, Emax=(31.19∓5.10)%, P<0.05]. Pretreatment of the rings with indomethacin abolished constrictions, and the relaxation was more obvious in endothelium-intact group [pD2=3.713∓0.11, Emax=(98.72∓0.34)%] than in endothelium- denuded group [pD2=3.54∓0.03, Emax=(94.56∓0.53)%, P<0.05]. CONCLUSION: Propofol induces constriction at low concentrations and relaxation at high concentrations in human intrapulmonary arteries with U46619-induced contraction. Indomethacin abolishes the constriction induced by propofol in isolated intrapulmonary arteries, suggesting that propofol potentiates U46619-mediated pulmonary vasoconstriction by promoting the concomitant production of prostaglandin by cyclooxygenase in pulmonary artery smooth muscle cells, and the mechanism for its relaxation effect may partly depend on the endothelium.

Role of tumour necrosis factor-a in the regulation of T-type calcium channel current in HL-1 cells.

Increasing evidence indicates that inflammation contributes to the initiation and perpetuation of atrial fibrillation (AF). Although tumour necrosis factor (TNF)-α levels are increased in patients with AF, the role of TNF-α in the pathogenesis of AF remains unclear. Besides L-type Ca(2+) currents (IC a,L ), T-type Ca(2+) currents (IC a,T ) also plays an important role in the pathogenesis of AF. This study was designed to use the whole-cell voltage-clamp technique and biochemical assays to explore if TNF-α is involved in the pathogenesis of AF through regulating IC a,T in atrial myocytes. It was found that compared with sinus rhythm (SR) controls, T-type calcium channel (TCC) subunit mRNA levels were decreased, while TNF-α expression levels were increased, in human atrial tissue from patients with AF. In murine atrial myocyte HL-1 cells, after culturing for 24 h, 12.5, 25 and 50 ng/mL TNF-α significantly reduced the protein expression levels of the TCC α1G subunit in a concentration-dependent manner. The peak current was reduced by the application of 12.5 or 25 ng/mL TNF-α in a concentration-dependent manner (from -15.08 ± 1.11 pA/pF in controls to -11.89 ± 0.83 pA/pF and -8.54 ± 1.55 pA/pF in 12.5 or 25 ng/mL TNF-α group respectively). TNF-α application also inhibited voltage-dependent inactivation of IC a,T, shifted the inactivation curve to the left. These results suggest that TNF-α is involved in the pathogenesis of AF, probably via decreasing IC a,T current density in atrium-derived myocytes through impaired channel function and down-regulation of channel protein expression. This pathway thus represents a potential pathogenic mechanism in AF.

Attenuation of microRNA-16 derepresses the cyclins D1, D2 and E1 to provoke cardiomyocyte hypertrophy.

Cyclins/retinoblastoma protein (pRb) pathway participates in cardiomyocyte hypertrophy. MicroRNAs (miRNAs), the endogenous small non-coding RNAs, were recognized to play significant roles in cardiac hypertrophy. But, it remains unknown whether cyclin/Rb pathway is modulated by miRNAs during cardiac hypertrophy. This study investigates the potential role of microRNA-16 (miR-16) in modulating cyclin/Rb pathway during cardiomyocyte hypertrophy. An animal model of hypertrophy was established in a rat with abdominal aortic constriction (AAC), and in a mouse with transverse aortic constriction (TAC) and in a mouse with subcutaneous injection of phenylephrine (PE) respectively. In addition, a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocyte and based on Ang-II-induced neonatal mouse ventricular cardiomyocyte respectively. We demonstrated that miR-16 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes in rats and mice. Overexpression of miR-16 suppressed rat cardiac hypertrophy and hypertrophic phenotype of cultured cardiomyocytes, and inhibition of miR-16 induced a hypertrophic phenotype in cardiomyocytes. Expressions of cyclins D1, D2 and E1, and the phosphorylated pRb were increased in hypertrophic myocardium and hypertrophic cardiomyocytes, but could be reversed by enforced expression of miR-16. Cyclins D1, D2 and E1, not pRb, were further validated to be modulated post-transcriptionally by miR-16. In addition, the signal transducer and activator of transcription-3 and c-Myc were activated during myocardial hypertrophy, and inhibitions of them prevented miR-16 attenuation. Therefore, attenuation of miR-16 provoke cardiomyocyte hypertrophy via derepressing the cyclins D1, D2 and E1, and activating cyclin/Rb pathway, revealing that miR-16 might be a target to manage cardiac hypertrophy.

Smad3 inactivation and MiR-29b upregulation mediate the effect of carvedilol on attenuating the acute myocardium infarction-induced myocardial fibrosis in rat.

Carvedilol, a nonselective ß-adrenoreceptor antagonist, protects against myocardial injury induced by acute myocardium infarction (AMI). The mechanisms underlying the anti-fibrotic effects of carvedilol are unknown. Recent studies have revealed the critical role of microRNAs (miRNAs) in a variety of cardiovascular diseases. This study investigated whether miR-29b is involved in the cardioprotective effect of carvedilol against AMI-induced myocardial fibrosis. Male SD rats were randomized into several groups: the sham surgery control, left anterior descending (LAD) surgery-AMI model, AMI plus low-dose carvedilol treatment (1 mg/kg per day, CAR-L), AMI plus medium-dose carvedilol treatment (5 mg/kg per day, CAR-M) and AMI plus high-dose carvedilol treatment (10 mg/kg per day, CAR-H). Cardiac remodeling and impaired heart function were observed 4 weeks after LAD surgery treatment; the observed cardiac remodeling, decreased ejection fraction, and fractional shortening were rescued in the CAR-M and CAR-H groups. The upregulated expression of Col1a1, Col3a1, and α-SMA mRNA was significantly reduced in the CAR-M and CAR-H groups. Moreover, the downregulated miR-29b was elevated in the CAR-M and CAR-H groups. The in vitro study showed that Col1a1, Col3a1, and α-SMA were downregulated and miR-29b was upregulated by carvedilol in a dose-dependent manner in rat cardiac fibroblasts. Inhibition of ROS-induced Smad3 activation by carvedilol resulted in downregulation of Col1a1, Col3a1, and α-SMA and upregulation of miR-29b derived from the miR-29b-2 precursor. Enforced expression of miR-29b significantly suppressed Col1a1, Col3a1, and α-SMA expression. Taken together, we found that smad3 inactivation and miR-29b upregulation contributed to the cardioprotective activity of carvedilol against AMI-induced myocardial fibrosis.

Fifty-Hertz electromagnetic fields facilitate the induction of rat bone mesenchymal stromal cells to differentiate into functional neurons.

BACKGROUND AIMS: Research results have shown that bone mesenchymal stromal cells (BMSC) can different into neural cells. Electromagnetic fields (EMF) play a role in regulating cell proliferation and differentiation, but the mechanisms behind this are unknown. In the present study, we explored the efficacy of EMF on the induction of rat BMSC differentiation into neurons in vitro. METHODS: First, rat BMSC were induced in a nerve cell culture environment and divided into three groups: an EMF induction treatment group (frequency of 50 Hz, magnetic induction of 5 mT, 60 min per day for 12 days), an induction-only group and a control group. Second, we observed cell phenotypes in a confocal microscope, tested gene expression through the use of reverse transcriptase-polymerase chain reaction, and detected postsynaptic currents by means of a cell patch-clamp. We analyzed the cell cycles and the portion of cells expressing neural cell markers with the use of flow cytometry. RESULTS: The results indicated that EMF can facilitate BMSC differentiation into neural cells, which expressed neuronal-specific markers and genes; they formed synaptic junctions and pulsed excitatory postsynaptic currents. At the same time, the G0-G1 phase ratio recorded by means of flow cytometry gradually decreased under the EMF treatment, whereas there was an increase of S-phase ratio, and the portion of cells expressing neuronal-specific markers increased. CONCLUSIONS: Given that a noninvasive treatment of 50-Hz EMF could significantly facilitate BMSC to differentiate into functional neurons, EMF appears to be a promising clinical option for stem cell transplantation therapies to combat central nervous system diseases.

MicroRNA 16 enhances differentiation of human bone marrow mesenchymal stem cells in a cardiac niche toward myogenic phenotypes in vitro.

AIM: Upregulation of microRNA 16 (miR-16) contributed to the differentiation of human bone marrow mesenchymal stem cells (hMSCs) toward myogenic phenotypes in a cardiac niche, the present study aimed to determine the role of miR-16 in this process. MAIN METHODS: hMSCs and neonatal rat ventricular myocytes were co-cultured indirectly in two chambers to set up a cardiac microenvironment (niche). miRNA expression profile in cardiac-niche-induced hMSCs was detected by miRNA microarray. Cardiac marker expression and cell cycle analysis were determined in different treatment hMSCs. Quantitative real-time PCR and Western blot were used to identify the expression of mRNA, mature miRNA and protein of interest. KEY FINDINGS: miRNA dysregulation was shown in hMSCs after cardiac niche induction. miR-16 was upregulated in cardiac-niche-induced hMSCs. Overexpression of miR-16 significantly increased G1-phase arrest of the cell cycle in hMSCs and enhanced the expression of cardiac marker genes, including GATA4, NK2-5, MEF2C and TNNI3. Differentiation-inducing factor 3 (DIF-3), a G0/G1 cell cycle arrest compound, was used to induce G1 phase arrest in cardiac-niche-induced hMSCs, and the expression of cardiac marker genes was up-regulated in DIF-3-treated hMSCs. The expression of CCND1, CCND2 and CDK6 was suppressed by miR-16 in hMSCs. CDK6, CCND1 or CCND2 knockdown resulted in G1 phase arrest in hMSCs and upregulation of cardiac marker gene expression in hMSCs in a cardiac niche. SIGNIFICANCE: miR-16 enhances G1 phase arrest in hMSCs, contributing to the differentiation of hMSCs toward myogenic phenotypes when in a cardiac niche. This mechanism provides a novel strategy for pre-modification of hMSCs before hMSC-based transplantation therapy for severe heart diseases.

Induced bone marrow mesenchymal stem cells improve cardiac performance of infarcted rat hearts.

We investigated whether transplantation of bone marrow mesenchymal stem cells (BMSC) with induced BMSC (iBMSC) or uninduced BMSC (uBMSC) into the myocardium could improve the performance of post-infarcted rat hearts. BMSCs were specified by flowcytometry. IBMSCs were cocultured with rat cardiomyocyte before transplantation. Cells were injected into borders of cardiac scar tissue 1 week after experimental infarction. Cardiac performance was evaluated by echocardiography at 1, 2, and 4 weeks after cellular or PBS injection. Langendorff working-heart and histological studies were performed 4 weeks after treatment. Myogenesis was detected by quantitative PCR and immunofluorescence. Echocardiography showed a nearly normal ejection fraction (EF) in iBMSC-treated rats and all sham control rats but a lower EF in all PBS-treated animals. The iBMSC-treated heart, assessed by echocardiography, improved fractional shortening compared with PBS-treated hearts. The coronary flow (CF) was decreased obviously in PBS and uBMSC-treated groups, but recovered in iBMSC-treated heart at 4 weeks (P < 0.01). Immunofluorescent microscopy revealed co-localization of Superparamagnetic iron oxide (SPIO)-labeled transplanted cells with cardiac markers for cardiomyocytes, indicating regeneration of damaged myocardium. These data provide strong evidence that iBMSC implantation is of more potential to improve infarcted cardiac performance than uBMSC treatment. It will open new promising therapeutic opportunities for patients with post-infarction heart failure.

[Expression and functional role of small conductance Ca(2+)-activated K(+) channels in human atrial myocytes].

OBJECTIVE: To investigate the expression and functional role of the small conductance Ca(2+)-activated K(+) channels in human atrial myocytes. METHODS: We collected the right atrial appendage tissues from 8 patients with congenital heart defect with sinus rhythm undergoing open-heart surgery. Immunohistochemistry was performed to identify the expression of 3 isoforms of SK channel (SK1, SK2 and SK3). Using the classical two-step enzymatic isolation method, perforated patch clamp and conventional voltage-clamp techniques were performed to record the action potentials (APs) and the whole-cell Ca(2+)-activated K(+) current (I(K, Ca)) in the single atrial myocyte. We compared the changes in action potential duration (APD) before and after the application of a specific SK channels blocker apamin (100 nmol/L). RESULTS: Human atrial myocytes showed positivity for all the SK1, SK2 and SK3 isoform channels. Patch-clamp recording confirmed the presence of I(K,Ca), and apamin significantly prolonged APD at 90% repolarization (APD(90)), but produced no obvious effect on APD(50). CONCLUSION: The three isoforms of SK channels are all expressed in human atrial myocytes. SK channels play a prominent role in the late phase of repolarization in human atrial myocytes, which is distinct from their functional roles in neurons where they mediate the process of afterhyperpolarization following APs.

Transcription factor Ap-1 mediates proangiogenic MIF expression in human endothelial cells exposed to Angiotensin II.

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with the atherosclerotic process and atherosclerotic plaque stability. MIF was shown to be highly expressed in advanced atherosclerotic lesions. Neutralizing MIF with a blocking antibody induced a regression of established atherosclerotic lesions. In this study, we investigated the mechanism underlying the proangiogenic effect of MIF in human umbilical vein endothelial cells (HUVECs). We showed that MIF induced the expression of angiogenesis-related genes in HUVECs. We also showed that MIF induced tube formation of HUVECs in vitro and in vivo. Angiotensin II (Ang II) could specifically up-regulate MIF expression in HUVECs. Using a luciferase reporter assay, we demonstrated that the AP-1 response element in the 5'-UTR of the MIF gene played a role in Ang II-induced MIF expression. Small hairpin RNA (shRNA) targeting c-Jun, a component of AP-1, and the AP-1 inhibitor CHX both efficiently inhibited MIF expression. The consistent result of electrophoretic mobility shift assay (EMSA) showed that Ang II specifically increased AP-1 activation in HUVECs. Our results suggest that AP-1 mediates Ang II-induced MIF expression which contributes to atherosclerotic plaque destabilization in human endothelial cells.

Improved liquid chromatography-tandem mass spectrometry method for the analysis of eptifibatide in human plasma.

A rapid, selective and highly sensitive high performance liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of eptifibatide in human plasma. Eptifibatide and the internal standard (IS), EPM-05, were extracted from plasma samples using solid phase extraction. Chromatographic separation was performed on a C(18) column at a flow rate of 0.5 mL/min. Detection of eptifibatide and the IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode. Traditional multiple reaction monitoring (MRM) using the transition of m/z 832.6-->m/z 646.4 and m/z 931.6-->m/z 159.4 was performed to quantify eptifibatide and the IS, respectively. The calibration curves were linear over the range of 1-1000 ng/mL with the lower limit of quantitation validated at 1 ng/mL. The intra- and inter-day precisions were within 13.3%, while the accuracy was within +/-7.6% of nominal values. The validated LC-MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of eptifibatide after intravenous (i.v.) administration of a 45 microg/kg bolus of eptifibatide to 8 healthy volunteers.

[Human bone marrow mesenchymal stem cells cocultured with semi-permeable membrane separated neonatal rat ventricular myocytes differentiated into cardiomyocyte phenotype].

OBJECTIVE: To investigate the ability of human bone marrow mesenchymal stem cells (hBMSCs), cocultured with semi-permeable membrane separated neonatal rat ventricular myocytes, to differentiate into cardiomyocytes. METHODS: hBMSCs were isolated and purified by density gradient centrifugation and adherence screening method. Cells were expanded as undifferentiated cells in culture for more than 3 passages and their phenotypes were identified with flow cytometer. hBMSCs were cocultured with neonatal rat ventricular myocytes in a rate of 1:10 separated by semi-permeable membrane. GATA4 mRNA was detected by RT-PCR; Immunocytochemistry, and Immunostaining were used to detect sarcomeric alpha-actinin, desmin, cTnT, and cTnI protein level. RESULTS: CD29 (98.64% +/- 0.80%) and CD44 (96.70% +/- 1.50%) were the major surface markers of hBMSCs. After coculturing with semi-permeable membrane separated neonatal rat ventricular myocytes, the first contraction of single cells was noted at day 7 and GATA4 expression was detected on these cells by RT-PCR after 1 to 3 weeks coculture. Desmin, sarcomeric alpha-actinin, cTnI and cTnT could be detected by immunocytochemistry and immunostaining on some of these cells. CONCLUSION: hBMSCs possess the potential to differentiate into myocardial cell phenotype in the cardiac microenvironment. Direct contact with cardiomyocytes was not necessary required for hBMSCs differentiation.

[Effect of adenoviral vectors on the differentiation potential of human bone marrow mesenchymal stem cells].

OBJECTIVE: To investigate the effect of adenoviral vector infection on the differentiation potential of human bone marrow mesenchymal stem cells (hMSCs). METHODS: The third-passage hMSCs were infected with the recombinant adenovirus expressing green fluorescence protein (GFP) for 2, 4, 8 and 16 days. RT-PCR was used to detect the mRNA expression of endodermal marker CYP 51, mesodermal marker SM22alpha, ectodermal marker nestin, pluripotent marker oct-4 and the alternative splicing factor nPTB. Seven days after adenovirus infection, the hMSCs were cultured in the presence of adipogenic agents for 14 days, and the adipose cells differentiated from hMSCs were detected with oil red O staining. RESULTS: Compared with normal hMSCs, the cell infected with the adenovirus for 2, 4, 8 and 16 days showed no obvious down-regulation of CYP51, SM22alpha, nestin, OCT4 or nPTB. The hMSCs 7 days after adenovirus infection were induced to differentiate into adipose cells, with a similar differentiation rate to that of normal hMSCs. CONCLUSION The differentiation potential of hMSCs is not affected by adenovirus infection, suggesting that adenovirus can be used as the gene delivery vector in MSC differentiation studies.

Noradrenaline release by activation of κ-bungarotoxin-sensitive nicotinic acetylcholine receptors participates in long-term potentiation-like response induced by nicotine.

Nicotine enhances the function of learning and memory, but the underlying mechanism still remains unclear. Hippocampal long-term potentiation (LTP) is assumed to be a cellular mechanism of learning and memory. Our previous experiments showed that with the single pulses evoking 80% of the maximal population spike (PS) amplitude, nicotine (10 µmol/L) induced LTP-like response in the hippocampal CA1 region. In the present study, the nicotinic acetylcholine receptor (nAChR) subtypes and relevant neurotransmitter releases involved in LTP-like response induced by nicotine were investigated by extracellularly recording the PS in the pyramidal cell layer in the hippocampal CA1 region in vitro. LTP-like response induced by nicotine was blocked by mecamylamine (1 µmol/L) or κ-bungarotoxin (0.1 µmol/L), but not by dihydro-ß-erythtroidine (DHßE, 10 µmol/L). Moreover, it was inhibited by propranolol (10 µmol/L), but not by phentolamine (10 µmol/L) or atropine (10 µmol/L). The results suggest that noradrenaline release secondary to the activation of κ-bungarotoxin-sensitive nAChRs participates in LTP-like response induced by nicotine in the hippocampal CA1 region.