[Analysis of protective effects of Chaihu Jia Longgu Muli Tang against ischemic stroke by combining traditional Chinese medicine pathogenesis and efficacy with modern pathology and pharmacology].

Chaihu Jia Longgu Muli Tang is a classical Chinese formulas treating Shaoyang syndrome complicated with Yangming syndrome according to Treatise on Febrile Diseases. This formula is used in mental disorder, nervous system, gynecologic, andrologic, and cardiovascular disease. However, its therapeutic effect on ischemia stroke and its mechanism is far from clear. In clinical practice, we have found that this formula is effective in treating ischemic stroke, which can shorten the course of the disease and reduce recurrence. The characteristics of this formula include: Shaoyang cardinal disadvantageous syndrome, mental and nervous symptoms, retained fluid punched upward syndrome and accumulation of heat in the stomach and intestines. By combining traditional Chinese medicine (TCM) pathogenesis and efficacy with modern pathology and pharmacology, we proposed that the TCM pathogenesis of stroke, which is characterized by hyperactivity of heat combining with phlegm, stasis and water drink, is consistent with syndromes and corresponding pathology targeted by Chaihu Jia Longgu Muli Tang, including the stress brain edema zone around the ischemic lesion, the increase of intracranial pressure, the excitement of sympathetic nerve, the release of monoamine neurotransmitter, the hypofunction of autonomic nervous system after stroke, and gastrointestinal stress response. Furthermore, the pharmacological mechanism of Chaihu Jia Longgu Muli Tang is concentrated on regulation the neuroendocrinology system centered by hypothalamic-pituitary-adrenal axis (HPA), participating in the process of neuron regeneration and apoptosis, oxidative stress, hyperactivity of sympathetic nerve, and inflammatory reaction. These pathological processes are consistent with the pathological changes after ischemic stroke. Therefore, Chaihu Jia Longgu Muli Tang is a key formula for treating ischemic stroke.

Inhibitory effects of hesperetin on Nav1.5 channels stably expressed in HEK 293 cells and on the voltage-gated cardiac sodium current in human atrial myocytes.

AIM: Voltage-gated sodium channels composed of a pore-forming α subunit and auxiliary ß subunits are responsible for the upstroke of the action potential in cardiac myocytes. The pore-forming subunit of the cardiac sodium channel Nav1.5, which is encoded by SCN5A, is the main ion channel that conducts the voltage-gated cardiac sodium current (INa) in cardiac cells. The current study sought to investigate the inhibitory effects of hesperetin on human cardiac Nav1.5 channels stably expressed in human embryonic kidney 293 (HEK 293) cells and on the voltage-gated cardiac sodium current (INa) in human atrial myocytes. METHODS: The effects of hesperetin on human cardiac Nav1.5 channels expressed in HEK 293 cells and on cardiac Na+ currents in human atrial myocytes were examined through whole-cell patch-clamp techniques. RESULTS: Nav1.5 currents were potently and reversibly suppressed in a concentration- and voltage-dependent manner by hesperetin, which exhibited an IC50 of 62.99 µmol/L. Hesperetin significantly and negatively shifted the voltage-dependent activation and inactivation curves. Hesperetin also markedly decelerated Nav1.5 current inactivation and slowed the recovery from Nav1.5 channel inactivation. The hesperetin-dependent blockage of Nav1.5 currents was frequency-dependent. Hesperetin also potently and reversibly inhibited Na+ current (INa) in human atrial myocytes, consistently with its effects on Nav1.5 currents in HEK 293 cells. CONCLUSION: Hesperetin is a potent inhibitor of INa in human atrial myocytes and Nav1.5 channels expressed in human embryonic kidney 293 cells. Hesperetin probably functions by blocking the open state and the inactivated state of these channels.

[Combined transgenic inhibition of CaMKII and Ik1 on cardiac remodeling].

This study was aimed to establish an experimental mouse model of combined transgenic inhibition of both multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and inward rectifier potassium current (Ik1), and to observe whether the specific inhibition of both CaMKII and Ik1 can bring about any effects on cardiac remodeling. Mice were divided into 4 groups: wild type (WT), CaMKII inhibited (AC3-I), Ik1 inhibited (Kir2.1-AAA) and combined inhibition of both CaMKII and Ik1 (AC3-I+Kir2.1-AAA). Mice in each group received electrocardiogram (ECG) and echocardiography examination. ECG in the condition of isoproterenol (ISO) injection was also checked. The whole cell patch clamp technique was used to measure Ik1 and the transient outward potassium current (Ito) from enzymatically isolated myocytes of left ventricle. In the condition of basal status, no significant changes of heart rate, PR interval and QRS interval were observed. No mouse showed ventricular arrhythmias in all of the 4 groups. After ISO injection, each group presented no significant ventricular arrhythmias either. The indexes measured by M-mode (motion-mode) and two-dimensional echocardiography had no significant differences among the four groups. Ik1 in AC3-I group was significantly higher than those in other three groups (P < 0.01) because of the results brought about by CaMKII inhibition. Among the latter three groups, both Kir2.1-AAA group and AC3-I+Kir2.1-AAA group had a significant reduced Ik1 compared with that of WT group, which was due to the Ik1 inhibition (P < 0.01). Ito in AC3-I group was higher than that of the other three groups (P < 0.01), but there were no significant differences in Ito among WT, Kir2.1-AAA and AC3-I+Kir2.1-AAA groups. Thus, combined transgenic myocardial CaMKII and Ik1 inhibition eliminated the up-regulation of Ik1 in CaMKII inhibited mice, and had no effects on cardiac remodeling including heart structure and function as well as arrhythmias at the basic and ISO conditions. The results of this study may provide a basis for the further investigation of combined inhibition of CaMKII and Ik1 in pathogenic cardiac remodeling.

M3 muscarinic acetylcholine receptor dysfunction inhibits Rac1 activity and disrupts VE-cadherin/ß-catenin and actin cytoskeleton interaction.

The objective was to investigate whether M3 muscarinic acetylcholine receptor (mAChR) dysfunction disrupts the linkage between the vascular endothelial (VE)-cadherin in the adherens junctional complex and the actin-based cytoskeleton, increasing vascular permeability in atherosclerosis. Western blotting revealed that a selective M3 receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and M3 receptor siRNA decrease VE-cadherin and ß-catenin in Triton X-100-insoluble fractions, indicating that M3 receptor inhibition weakens the linkage between the VE-cadherin/ß-catenin complex and the actin cytoskeleton. Co-immunoprecipitation assays showed that M3 receptor inhibition reduces Rac1 activity and the association of IQ motif-containing GTPase-activating protein 1 (IQGAP1) with Ras-related C3 botulinum toxin substrate 1 (Rac1), while increasing the interaction between IQGAP1 and ß-catenin. Using IQGAP1 siRNA, we found that IQGAP1 is required for stable interaction between VE-cadherin/ß-catenin and the actin cytoskeleton in quiescent endothelial cells; IQGAP1 siRNA augments the M3 receptor inhibition-induced dissociation between them. Moreover, S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, attenuates this disassociation and Rac1 activity inhibition. The M3 receptor facilitates interaction of the VE-cadherin-based adherens junctional complex and the actin-based cytoskeleton by maintaining Rac1 activity, which regulates the interaction between IQGAP1/Rac1 and IQGAP1/ß-catenin, and may contribute to endothelial barrier function under physiological conditions.

Evidence for functional expression of TRPM7 channels in human atrial myocytes.

Transient receptor potential melastatin-7 (TRPM7) channels have been recently reported in human atrial fibroblasts and are believed to mediate fibrogenesis in human atrial fibrillation. The present study investigates whether TRPM7 channels are expressed in human atrial myocytes using whole-cell patch voltage-clamp, RT-PCR and Western blotting analysis. It was found that a gradually activated TRPM7-like current was recorded with a K(+)- and Mg(2+)-free pipette solution in human atrial myocytes. The current was enhanced by removing extracellular Ca(2+) and Mg(2+), and the current increase could be inhibited by Ni(2+) or Ba(2+). The TRPM7-like current was potentiated by acidic pH and inhibited by La(3+) and 2-aminoethoxydiphenyl borate. In addition, Ca(2+)-activated TRPM4-like current was recorded in human atrial myocytes with the addition of the Ca(2+) ionophore A23187 in bath solution. RT-PCR and Western immunoblot analysis revealed that in addition to TRPM4, TRPM7 channel current, mRNA and protein expression were evident in human atrial myocytes. Interestingly, TRPM7 channel protein, but not TRPM4 channel protein, was significantly increased in human atrial specimens from the patients with atrial fibrillation. Our results demonstrate for the first time that functional TRPM7 channels are present in human atrial myocytes, and the channel expression is upregulated in the atria with atrial fibrillation.

Role of Neuroimmune Interactions in COVID-19-related Cardiovascular Damage.

Coronavirus disease 2019 (COVID-19) has caused a global pandemic impacting over 200 countries/regions and more than 200 million patients worldwide. Among the infected patients, there is a high prevalence of COVID-19-related cardiovascular injuries. However, the specific mechanisms linking cardiovascular damage and COVID-19 remain unclear. The COVID-19 pandemic also has exacerbated the mental health burden of humans. Considering the close association between neuroimmune interactions and cardiovascular disease, this review assessed the complex pathophysiological mechanisms connecting neuroimmune interactions and cardiovascular disease. It was revealed that the mental health burden might be a pivotal accomplice causing COVID-19-associated cardiovascular damage. Specifically, the proinflammatory status of patients with a terrible mood state is closely related to overdrive of the hypothalamus-pituitary-adrenal (HPA) axis, sympathovagal imbalance, and endothelial dysfunction, which lead to an increased risk of developing cardiovascular injury during COVID-19. Therefore, during the prevention and treatment of cardiovascular complications in COVID-19 patients, particular attention should be given to relieve the mental health burden of these patients.

Clinical risk score for postoperative pneumonia following heart valve surgery.

BACKGROUND: Postoperative pneumonia (POP) is one of the most common infections following heart valve surgery (HVS) and is associated with a significant increase in morbidity, mortality, and health care costs. This study aimed to identify the major risk factors associated with the occurrence of POP following HVS and to derive and validate a clinical risk score. METHODS: Adults undergoing open HVS between January 2016 and December 2019 at a single institution were enrolled in this study. Patients were randomly assigned to the derivation and validation sets at 1:1 ratio. A prediction model was developed with multivariable logistic regression analysis in the derivation set. Points were assigned to independent risk factors based on their regression coefficients. RESULTS: POP occurred in 316 of the 3853 patients (8.2%). Multivariable analysis identified ten significant predictors for POP in the derivation set, including older age, smoking history, chronic obstructive pulmonary disease, diabetes mellitus, renal insufficiency, poor cardiac function, heart surgery history, longer cardiopulmonary bypass, blood transfusion, and concomitant coronary and/or aortic surgery. A 22-point risk score based on the multivariable model was then generated, demonstrating good discrimination (C-statistic: 0.81), and calibration (Hosmer-Lemeshow χ2 = 8.234, P = 0.312). The prediction rule also showed adequate discriminative power (C-statistic: 0.83) and calibration (Hosmer-Lemeshow χ2 = 5.606, P = 0.691) in the validation set. Three risk intervals were defined as low-, medium-, and high-risk groups. CONCLUSION: We derived and validated a 22-point risk score for POP following HVS, which may be useful in preventive interventions and risk management. TRIAL REGISTRATION: Chictr.org, ChiCTR1900028127; http://www.chictr.org.cn/showproj.aspx?proj=46932.

Functional ion channels in mouse cardiac c-kit(+) cells.

Cardiac c-kit(+) cells are generally believed to be the major population of stem/progenitor cells in the heart and can be used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not understood in this type of cells. The present study was designed to investigate functional ion channels in undifferentiated mouse cardiac c-kit(+) cells using approaches of whole cell patch voltage clamp, RT-PCR, and cell proliferation assay. It was found that three types of ionic currents were present in mouse cardiac c-kit(+) cells, including a delayed rectifier K(+) current (IK(DR)) inhibited by 4-aminopyridine (4-AP), an inward rectifier K(+) current (I(Kir)) decreased by Ba(2+), and a volume-sensitive chloride current (I(Cl.vol)) inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB). RT-PCR revealed that the corresponding ion channel genes, Kv1.1, Kv1.2, and Kv1.6 (for IK(DR)), Kir.1.1, Kir2.1, and Kir2.2 (likely responsible for I(Kir)), and Clcn3 (for I(Cl.vol)), were significant in mouse cardiac c-kit(+) cells. The inhibition of I(Cl.vol) with NPPB and niflumic acid, but not IK(DR) with 4-AP and tetraethylammonium, reduced cell proliferation and accumulated the cell progression at G(0)/G(1) phase in mouse cardiac c-kit(+) cells. Our results demonstrate that three types of functional ion channel currents (i.e., IK(DR), I(Kir), and I(Cl.vol)) are present in mouse cardiac c-kit(+) cells, and I(Cl.vol) participates in regulating cell proliferation.

Acacetin, a natural flavone, selectively inhibits human atrial repolarization potassium currents and prevents atrial fibrillation in dogs.

BACKGROUND: The development of atrium-selective antiarrhythmic agents is a current strategy for inhibiting atrial fibrillation (AF). The present study investigated whether the natural flavone acacetin from the traditional Chinese medicine Xuelianhua would be an atrium-selective anti-AF agent. METHODS AND RESULTS: The effects of acacetin on human atrial ultrarapid delayed rectifier K(+) current (I(Kur)) and other cardiac ionic currents were studied with a whole-cell patch technique. Acacetin suppressed I(Kur) and the transient outward K(+) current (IC(50) 3.2 and 9.2 mumol/L, respectively) and prolonged action potential duration in human atrial myocytes. The compound blocked the acetylcholine-activated K(+) current; however, it had no effect on the Na(+) current, L-type Ca(2+) current, or inward-rectifier K(+) current in guinea pig cardiac myocytes. Although acacetin caused a weak reduction in the hERG and hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells, it did not prolong the corrected QT interval in rabbit hearts. In anesthetized dogs, acacetin (5 mg/kg) prolonged the atrial effective refractory period in both the right and left atria 1 to 4 hours after intraduodenal administration without prolongation of the corrected QT interval, whereas sotalol at 5 mg/kg prolonged both the atrial effective refractory period and the corrected QT interval. Acacetin prevented AF induction at doses of 2.5 mg/kg (50%), 5 mg/kg (85.7%), and 10 mg/kg (85.7%). Sotalol 5 mg/kg also prevented AF induction (60%). CONCLUSIONS: The present study demonstrates that the natural compound acacetin is an atrium-selective agent that prolongs the atrial effective refractory period without prolonging the corrected QT interval and effectively prevents AF in anesthetized dogs after intraduodenal administration. These results indicate that oral acacetin is a promising atrium-selective agent for the treatment of AF.

Rapamycin Treatment Attenuates Angiotensin II -induced Abdominal Aortic Aneurysm Formation via VSMC Phenotypic Modulation and Down-regulation of ERK1/2 Activity.

The aim of the present study is to address the effect of rapamycin on abdominal aortic aneurysm (AAA) and the potential mechanisms. A clinically relevant AAA model was induced in apolipoprotein E-deficient (ApoE-/-) mice, in which miniosmotic pump was implanted subcutaneously to deliver angiotensin II (Ang II) for 14 days. Male ApoE-/- mice were randomly divided into 3 groups: saline infusion, Ang II infusion, and Ang II infusion plus intraperitoneal injection of rapamycin. The diameter of the supra-renal abdominal aorta was measured by ultrasonography at the end of the infusion. Then aortic tissue was excised and examined by Western blotting and histoimmunochemistry. Ang n with or without rapamycin treatment was applied to the cultured vascular smooth muscle cells (VSMCs) in vitro. The results revealed that rapamycin treatment significantly attenuated the incidence of Ang II induced-AAA in ApoE-/- mice. Histologic analysis showed that rapamycin treatment decreased disarray of elastin fibers and VSMCs hyperplasia in the medial layer. Immunochemistry staining and Western blotting documented the increased phospho-ERK1/2 and ERK1/2 expression in aortic walls in Ang II induced-AAA, as well as in human lesions. Whereas in the rapamycintreated group, decreased phospho-ERKl/2 expression level was detected. Moreover, rapamycin reversed Ang II -induced VSMCs phenotypic change both in vivo and in vitro. Based on those results, we confirmed that rapamycin therapy suppressed Ang II -induced AAA formation in mice, partially via VSMCs phenotypic modulation and down-regulation of ERK1/2 activity.

[An experimental study of recombinant human erythropoietin on the treatment of acute myocardial infarction in rats].

OBJECTIVE: To observe the treatment and it's mechanisms of rHu-EPO on acute myocardial infarction of SD rats in vitro and vivo. METHODS: Cardiomyocytes were isolated from neonatal Sprague-Dawley rats. Hypoxia condition and oxidative stress were used to induce apoptosis. rHu-EPO was added to the culture system. Apoptosis was assessed by using Hoechst 33258 dyeing. Apoptosis index (AI) was then calculated. Thirty two rats were divided into three groups including sham operation group (Sham), acute myocardial infarction group (MI) and treated group (MI + EPO). Acute myocardial infarction model was made by ligating the anterior descending coronary artery. rHu-EPO was administered i.p. in MI + EPO group at the dose of 5000 units/kg of body weight immediately after the ligation and the next six days. At the fourteenth day all animals underwent hemodynamic measurements and then executed, the samples were examined with hematoxylin and eosin (HE) stain, immunohistochemistry technique (Bcl-2, Bax) and TdT-mediated dUTP nick end labeling (TUNEL) dyeing. RESULTS: rHu-EPO significantly down-regulated the apoptosis of cardiomyocytes which underwent hypoxia or oxidative stress. In vivo experiment rHu-EPO protected the hemodynamic function of the rats from myocardial infarction and down-regulated the ratio of the positive cells for TUNEL and Bax. The ratio of the positive cells for Bcl-2 was up-regulated by rHu-EPO. CONCLUSION: These findings suggested rHu-EPO improve myocardial infarction by attenuating apoptosis. Potential mechanism is to up-regulated Bcl-2 expression and down-regulated Bax expression.

Protein kinase C enhances the swelling-induced chloride current in human atrial myocytes.

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

Nicotinic α7 receptor inhibits the acylation stimulating protein­induced production of monocyte chemoattractant protein­1 and keratinocyte­derived chemokine in adipocytes by modulating the p38 kinase and nuclear factor­κB signaling pathways.

Obesity is associated with chronic low­grade inflammation, which is characterized by increased infiltration of macrophages into adipose tissue. Acylation stimulating protein (ASP) is an adipokine derived from the immune complement system, which constitutes a link between adipocytes and macrophages, and is involved in energy homeostasis and inflammation. The purpose of the present study was to preliminarily investigate in vitro, whether functional α7nAChR in adipocytes may suppress ASP­induced inflammation and determine the possible signaling mechanism. Studies have reported associations between the expression of α7 nicotinic acetylcholine receptor (α7nAChR) and obesity, insulin resistance and diabetes. Additionally, α7nAChRs are important peripheral mediators of chronic inflammation, which is a key contributor to health problems in obesity. The primary aim of the present study was to evaluate the impact of exogenous ASP and α7nAChR on macrophage infiltration in adipose tissue and to examine the potential underlying molecular mechanism. Western blot analysis revealed that recombinant ASP increased the expression levels of monocyte chemoattractant protein­1 (MCP­1) and keratinocyte­derived chemokine (KC) by 3T3­L1 adipocytes. However, nicotine significantly inhibited the production of ASP­induced cytokines via the stimulation of α7nAChR. It was also found that α7nAChR inhibited the ASP­induced activation of p38 kinase and nuclear factor­κB (NF­κB), and the production of MCP­1 and KC. These data indicated that α7nAChR caused the inhibition of ASP­induced activation of p38 kinase and NF­κB to inhibit the production of MCP­1 and KC.

Inhibitory effects of hesperetin on Kv1.5 potassium channels stably expressed in HEK 293 cells and ultra-rapid delayed rectifier K(+) current in human atrial myocytes.

In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K(+) current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 µΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300µM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30µM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30µM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K(+) current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell.

Distinctive property and pharmacology of voltage-gated sodium current in rat atrial vs ventricular myocytes.

BACKGROUND: Several mammalian species display distinct biophysical properties between atrial and ventricular voltage-gated sodium current (INa); however, the potential mechanism behind this phenomenon is unknown. OBJECTIVE: The purpose of this study was to investigate the potential molecular identities of the different INa in atrial and ventricular myocytes of rat hearts. METHODS: Whole-cell patch voltage-clamp and molecular biology techniques were used in the study. RESULTS: Ventricular INa exhibited a slower inactivation, more positive potential of inactivation, and quicker recovery from inactivation compared to atrial INa. Real-time polymerase chain reaction and western blot analysis revealed that mRNA and protein levels of NaVß2 and NaVß4 subunits, but not NaV1.5, were greater in ventricular myocytes than in atrial myocytes. INa in heterologous HEK 293 cell expression system with coexpressing hNaV1.5 and hNaVß2/hNaVß4 showed similar biophysical properties to ventricular INa. Greater protein expression of NaVß2 and NaVß4 subunits was also observed in human ventricles. Interestingly, pharmacologic study revealed that the antiarrhythmic drug dronedarone (10 µM) inhibited atrial INa more (by 73%) than ventricular INa (by 42%), and shifted its inactivation to more negative voltages (-4.6 mV) compared to ventricular INa. CONCLUSION: The results of this study demonstrate the novel information that the distinctive biophysical properties of INa in atrial and ventricular myocytes can be attributed to inhomogeneous expression of NaVß2 and NaVß4 subunits, and that atrial INa is more sensitive to inhibition by dronedarone.

Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases.

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (I(Cl.vol)) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO(4)(-3)). I(Cl.vol) evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC(50) = 22.4 microM); 100 microM genistein stimulated I(Cl.vol) by 122.4 +/- 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 150 microM) and tamoxifen (20 microM), blockers of I(Cl.vol). Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl(-) current in 1T. In contrast to the stimulatory effects of genistein, 100 microM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I(Cl.vol) by 38.2 +/- 4.9% and 40.9 +/- 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter I(Cl.vol). In addition, the PTP inhibitor VO(4)(-3) (1 mM) reduced I(Cl.vol) by 53.5 +/- 4.5% (IC(50) = 249.6 microM). Pretreatment with VO(4)(-3) antagonized genistein-induced augmentation and A23- or A25-induced suppression of I(Cl.vol). Furthermore, the selective Src-family PTK inhibitor PP2 (5 microM) stimulated I(Cl.vol), mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 microM) reduced I(Cl.vol), mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO(4)(-3). The results suggest that I(Cl.vol) is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on I(Cl.vol), and multiple target proteins are likely to be involved.

[Heat-shock protein 70 may be a putative endogenous ligand of Toll-like receptor-4 of human monocytes].

OBJECTIVE: To explore the relation between human heat shock protein 70 (HSP70) and Toll-like receptor-4 (TLR4) in human monocytes. METHODS: Periphery blood mononuclear cells were isolated from the samples of healthy blood donors' whole blood and monocytes were prepared and cultured. HSP70 of the final concentrations of 2.5 microg/ml, 5.0 microg/ml, 7.5 microg/ml, and 10 microg/ml respectively was added; 6 hours later the concentration of TNF-alpha in the supernatant was detected. Another monocytes were cultured and HSP70 of the final concentration of 5.0 microg/ml was added and the concentrations of NF-kappaB were detected 0, 30, 60, and 120 minutes later respectively. TLR4 blocker of the final concentrations of 5 microg/ml, 20 microg/ml, and 30 microg/ml respectively was added into another culture for 30 minutes and 5.0 microg/ml HSP70 was added, then immunochemistry was used to detect the concentration of NF-kappaB 120 minutes after ELISA was used to detect the concentration of and TNF-alpha 8 hours later. In order to examine the influence of HSP70 on the TLR4 in the cytomembrane of monocytes, HSP70 of the final concentration of 5.0 microg/ml was added into the culture of monocytes for 0, 30, 60, and 120 minutes respectively then flow cytometry was used to detect the mean fluorescence intensity (MFI) of TLR4. RESULTS: HSP70 stimulation increased the TNF-a concentration in the supernatant dose-dependently. The percentages of NF-kappaB positive monocytes were 38 +/- 6, 67 +/- 12, and 54 +/- 12 30 min, 60 min, 120 min after HSP70 stimulation, all significantly higher than that at the beginning of experiment (17 +/- 6, P < 0.05, P < 0.01, and P < 0.01). The percentages of NF-kappaB positive monocytes were 39% +/- 4%, 32% +/- 6%, and 28% +/- 6% 120 minutes after anti-TLR4 mAb stimulation, all significantly lower than that of the control group (67% +/- 12%, all P < 0.05). TLR4 blocker of different concentrations significantly inhibited the TNF-alpha secretion by the monocytes (all P < 0.05). The MFI of TLR4 in the cytomembrane of monocyte was significantly down-regulated 60 minutes, especially 120 minutes, after the HSP70 stimulation in comparison with that before the stimulation (P < 0.05 and P < 0.01). CONCLUSION: TLR4 appears to be involved in HSP70-mediated activation of innate immunity.

Calcium-activated transient outward chloride current and phase 1 repolarization of swine ventricular action potential.

OBJECTIVE: It is unknown whether 4-aminopyridine- (4-AP-) sensitive transient outward K(+) current (I(to1)) and/or Ca(2+)-activated transient outward Cl(-) current (I(Ca.Cl) or I(to2)) contribute(s) to phase 1 repolarization of pig ventricular action potential (AP). The purpose of the present study was to determine ionic contribution of the phase 1 repolarization of AP in pig ventricle. METHODS: We used whole-cell patch techniques to record APs and membrane currents, and Western immunoblotting analysis to detect expression of I(to1) protein (Kv4.2 or Kv4.3) in pig ventricular myocytes. RESULTS: A transient outward current (I(to)) was activated upon depolarization voltage steps to between -10 and +60 mV from -50 mV in pig ventricular cells, and the I(to) was resistant to 4-AP application, but sensitive to the inhibition by ryanodine (10 micromol/l) and the Ca(2+) channel blockade, and the Cl(-) channel blocker 4,4'-diisothiocyanostilben-2,2'disulfonic acid (DIDS, 150 micromol/l). The current was diminished by external Cl(-) (Cl(-)(o)) replacement and showed a 'bell-shaped' I-V relationship at room temperature, typical of I(to2). No difference in I(to2) was observed in the regional cells from epicardium, midmyocardium, and endocardium of left ventricle. APs showed significant phase 1 and 'spike and dome' in pig ventricular myocytes. The phase 1 and 'spike and dome' of APs were not affected by 4-AP (3 mmol/l), but abolished by replacing Cl(-)(o) and by application of 100 micromol/l DIDS, suggesting I(to2) contribution. Western immunoblotting analysis showed no evidence for the expression of 4-AP-sensitive I(to1) channel protein (Kv4.2 or Kv4.3) in pig ventricular cells. CONCLUSION: The results indicate that 4-AP-sensitive I(to1) is not expressed, and only Ca(2+)-activated I(to2) is present in pig cardiac cells, which contributes importantly to the phase 1 repolarization of ventricular APs in this species.

Biventricular repair versus uni-ventricular repair for pulmonary atresia with intact ventrical septum: A systematic review.

The management of pulmonary atresia with intact ventricular septum (PA/IVS) remains controversial. The goal of separating systematic and pulmonary circulation can be achieved by biventricular or uni-ventricular (Fontan or one and a half ventricle repair) strategies. Although outcomes have been improved, these surgical procedures are still associated with high mortality and morbidity. An optimal strategy for definitive repair has yet to be defined. We searched databases for genetically randomized controlled trials (RCTs) comparing biventricular with uni-ventricular repair for patient with PA/IVS. Data extraction and quality assessment were performed following the guidelines of the Cochrane Collaboration. Primary outcome measures were overall survival, and secondary criteria included exercise function, arrhythmia-free survival and treatment-related mortality. A total number of 669 primary citations were screened for relevant studies. Detailed analysis revealed that no RCTs were found to adequately address the research question and no systematic meta-analysis would have been carried out. Nevertheless, several retrospective analyses and case series addressed the question of finding right balance between biventricular and uni-ventricular repair for patient with PA/IVS. In this review, we will discuss the currently available data.

Type 3 muscarinic acetylcholine receptor stimulation is a determinant of endothelial barrier function and adherens junctions integrity: role of protein-tyrosine phosphatase 1B.

The main purpose of this study was to investigate whether type 3 muscarinic acetylcholine receptor (M3R) dysfunction induced vascular hyperpermeability. Transwell system analysis showed that M3R inhibition by selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and small interfering RNA both increased endothelial permeability. Using coimmunoprecipitation and Western blot assay, we found that M3R inhibition increased VE-cadherin and ß-catenin tyrosine phosphorylation without affecting their expression. Using PTP1B siRNA, we found that PTP1B was required for maintaining VE-cadherin and ß-catenin protein dephosphorylation. In addition, 4-DAMP suppressed PTP1B activity by reducing cyclic adenosine monophosphate (cAMP), but not protein kinase Cα (PKCα). These data indicate that M3R preserves the endothelial barrier function through a mechanism potentially maintaining PTP1B activity, keeping the adherens junction proteins (AJPs) dephosphorylation.