BACKGROUND: Short QT (SQT) syndrome (SQT) 1 is an inherited sudden death syndrome often associated with atrial fibrillation (AF). We examined the cellular basis for AF in a newly developed experimental atrial model of SQT1. METHODS: Action potentials (APs) were recorded from the pectinate muscle (PM) and crista terminalis (CT) regions of coronary-perfused canine right atrial preparations, together with a pseudoelectrocardiogram. The I(Kr) agonist PD-118057 (20 muM) was used to mimic the gain of function in I(Kr) known to underlie SQT1. RESULTS: The I(Kr) agonist significantly abbreviated the AP duration (APD) of CT and PM and of the effective refractory period (ERP) measured in PM (n = 28). Spatial dispersion of repolarization (SDR), defined as inter-regional differences of APD, increased from 27 +/- 17 ms to 51 +/- 32 ms (P = .002; n = 28). AF could be induced by a single premature stimulus after but not before exposure to PD-118057 in 26/28 (93%) preparations. Quinidine (10 muM), which prolonged APD and ERP, but not lidocaine (20 muM) or E-4031 (5 muM), which prolonged only ERP or APD, respectively, was effective in preventing the PD-118057-mediated AF. In the presence of PD-118057, isoproterenol (100 nM) further abbreviated both APD and ERP and facilitated induction of sustained AF in five of six preparations. CONCLUSIONS: The I(Kr) agonist recapitulates the electrophysiologic and arrhythmic manifestations of SQT1. Abbreviation of APD and ERP and amplification of SDR predispose to the development of AF by creating the substrate for reentry. Quinidine, but not E-4031 or lidocaine, was effective in preventing AF in this setting. less...

Therapies to correct the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) folding defect require sensitive methods to detect channel activity in vivo. beta2 adrenergic receptor agonists, the CFTR stimuli commonly used in nasal potential difference assays, may not overcome the channel gating defects seen in DeltaF508 CFTR following plasma membrane localization. In this study we identify an agent, quercetin, that enhances detection of surface DeltaF508 CFTR, and is suitable for nasal perfusion. A screen of flavonoids in CFBE41o- cells stably transduced with DeltaF508 CFTR, corrected to the cell surface with low temperature growth, revealed that quercetin stimulated an increase in short-circuit current. This increase was dose-dependent in both fisher rat thyroid and CFBE41o- cells; high concentrations inhibited Cl(-) conductance. In CFBE41o- airway cells, quercetin (20 mug/ml) activated DeltaF508 CFTR, where beta2 adrenergic receptor agonist isoproterenol did not. Quercetin had small effects on cAMP levels, but did not produce detectable phosphorylation of isolated CFTR R-domain, suggesting activation independent of channel phorphorylation. When perfused in the nares of Cftr + mice, quercetin (20 microg/ml) produced hyperpolarization of potential difference that was absent in Cftr -/- mice. Finally, quercetin-induced dose-dependent hyperpolarization of nasal potential difference was also seen in normal human subjects. Quercetin activates CFTR mediated anion transport in respiratory epithelia in vitro and in vivo, and may be useful in studies intended to detect rescue of DeltaF508 CFTR by nasal potential difference. less...

The vascular system secretes many bioactive factors. In a gene chip database, we searched for novel genes with signal sequences that are specifically expressed in murine aorta, and focused on one gene previously named CCDC3 (NCBI nucleotide entry NM_028804). Northern blot analysis revealed that CCDC3 was expressed abundantly in the aorta and adipose tissues. The mRNA levels of CCDC3 were higher in adipose tissues of obese db/db mice than control mice, and induced during differentiation of rat primary adipocytes. In differentiated adipocytes, CCDC3 mRNA expression was enhanced by insulin and pioglitazone, a PPARgamma agonist, and suppressed by TNF-alpha, isoproterenol and norepinephrine. Transient expression experiments followed by N-terminal amino acid sequence analysis revealed secretion of CCDC3 protein into the culture medium, which was dose-dependently reduced by brefeldin A, an inhibitor of Golgi-mediatedsecretory pathway. When expressed in COS-7 cells, CCDC3 protein was post-transcriptionally modified with N-glycosylation, and formed a dimer complex. These results indicate that CCDC3 is a protein secreted by adipocytes and endothelial cells, and that its level is regulated both hormonally and nutritionally. less...

Mutations in the lamin A/C gene (LMNA) cause lipodystrophy. However, little data are available on lamin A/C expression in various fat depots in women We recruited 34 women scheduled for gynecologic surgery. Blood samples were collected on the morning of surgery to obtain a detailed lipid profile. Radiological examinations were performed to measure total body fat mass and abdominal fat accumulation. Fat samples were taken from the subcutaneous (SC) fat depot and from the greater omentum (OM) during the surgical procedure. Whole adipose tissue samples were used for total messenger RNA (mRNA) extraction and real-time polymerase chain reaction quantification of the LMNA transcript. No association was observed between lamin A/C mRNA expression, either in SC or OM fat tissue, and adiposity measures Women with low SC lamin A/C expression, identified on the basis of the median value of SC lamin A/C mRNA expression, had a significantly altered lipid profile including lower levels of high-density lipoprotein (HDL) cholesterol and HDL(2) cholesterol and reduced HDL(2) cholesterol to HDL(3) cholesterol ratio (P < .05 for all) These women were also characterized by higher cholesterol to HDL cholesterol, low-density lipoprotein-triglycerides, very low-density lipoprotein-apolipoprotein B, and low-density lipoprotein cholesterol to HDL cholesterol (P < .05 for all). Low SC lamin A/C mRNA expression levels were also associated with significantly increased lipolysis in isolated fat cells from this fat depot Specifically, the response to lipolytic agent isoproterenol was significantly increased at doses ranging from 10(-5) to 10(-10) mol/L (P < .05). A similar trend was observed in OM fat cells but did not reach significance. In conclusion, low lamin A/C expression in SC adipose tissue is associated with significant alterations in the lipid profile and increased fat cell lipolysis, independent of the level of total or abdominal adiposity less...

Membrane rafts and caveolae are specialized microdomains of the cell membrane that form physical platforms for compartmentalization of signalling molecules. Here, we intended to gain insight into the consequences of caveolar localization in G protein-coupled receptor function. We analysed beta(2)-adrenoceptor signalling in purified CRLDF (caveolin-rich low density fractions) of beta(2)-adrenoceptor-overexpressing HEK-293 cells. beta(2)-adrenoceptor and Gs immunoreactivities and forskolin-stimulated adenylate cyclase activity were all detected in CRLDF obtained by the conventional raft purification method that uses Triton X-100 solubilization. However, Triton X-100 caused a complete loss of the functional coupling between beta(2)-adrenoceptor, Gs and adenylate cyclase. Therefore, we developed an optimized purification method based on n-octyl-beta-D-glucopyranoside solubilization, where the functional properties of beta(2)-adrenoceptor, Gs and adenylate cyclase were preserved in the CRLDF. Using this method, we showed that isoproterenol-stimulated adenylate cyclase activity was similar in CRLDF and bulk membrane preparations of HEK-293 cells that overexpress beta(2)-adrenoceptor or beta(2)-adrenoceptor-Gs fusion. Accordingly, treatment of cells with methyl-beta-cyclodextrin, a caveola-disrupting agent, did not affect beta(2)-adrenoceptor-induced cAMP response. Likewise, these responses were insensitive to caveolin 1 and 2 overexpression. On the other hand, methyl-beta-cyclodextrin treatment did decrease beta(2)-adrenoceptor-induced ERK phosphorylation. However, the latter effect of methyl-beta-cyclodextrin could be attributed to a non-specific effect rather than its ability to disrupt membrane microdomains. We showed that localization in the raft microdomains did not affect the signalling efficiency of beta(2)-adrenoceptor - Gs - adenylate cyclase pathway, and that methyl-beta-cyclodextrin may inhibit signalling by directly affecting the signalling system independently of its caveola-disrupting property. less...

Myocardial infarction affects a large population in the world. Lipid peroxide metabolism plays an important role in the pathology of myocardial infarction. This study aims to evaluate the preventive effect of caffeic acid on lipid peroxides, antioxidants, cardiac marker enzymes, and histopathological findings in isoproterenol (ISO)-induced myocardial-infarcted male Wistar rats. Myocardial infarction was induced in rats by subcutaneous injection of ISO (100 mg/kg) at an interval of 24 hours for 2 days. The ISO-induced rats showed significant increase in the levels of thiobarbituric acid reactive substances, lipid hydroperoxides in the heart, plasma uric acid, and serum cardiac marker enzymes, and significant decrease in the activities of heart superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, and the levels of reduced glutathione, vitamin E, and vitamin C in the plasma and heart. Oral pretreatment with caffeic acid (15 mg/kg) daily for 10 days showed significant decrease in the levels of serum cardiac marker enzymes, heart lipid peroxidation products and plasma uric acid and significant increase in the levels of antioxidant system. Histopathology of myocardium also confirmed the protective effect of caffeic acid in myocardial-infarcted rats. In vitro study on total antioxidant activity (2,2'-azinobis-[3-ethylbenzothiazoline-6-sulfonic acid](+) assay) confirmed the strong antioxidant action of caffeic acid. Thus, the present study revealed that caffeic acid ameliorates cardiac damage in ISO-induced myocardial infarction by maintaining lipid peroxide metabolism due to its free radical scavenging and antioxidant effects. A diet containing caffeic acid may be beneficial to myocardial infarction. less...

OBJECTIVE: It has been shown that Ang-(1-7) has cardioprotective actions. To directly investigate the effects of Ang-(1-7) specifically in the heart, we generated and characterized transgenic (TG) rats which express an Ang-(1-7)-producing fusion protein driven by the alpha-MHC promoter. METHODS: and RESULTS: After microinjection of the transgene into fertilized rat zygotes, we obtained four different transgenic lines. Homozygous animals were analyzed with regard to the expression profile of the transgene by ribonuclease protection assay. Transgene expression was detected mainly in the heart with weak or no expression in other organs. Heterozygous TG(hA-1-7)L7301 rats presented a significant increase in cardiac Ang-(1-7) concentration compared with control rats (17.1 +/- 2.1 versus 3.9 +/- 1.4 pg/mg protein in SD rats). Radiotelemetry analysis revealed that TG rats presented no significant changes in blood pressure and heart rate compared with normal rats. Overexpression of Ang-(1-7) in the heart produced slight improvement in resting cardiac function (+ dT/dt: 81530 +/- 1305.0 versus 77470 +/- 345.5 g/s bpm in SD rats, p < 0.05), which was in keeping with the enhanced [Ca(2+)] handling observed in cardiomyocytes of TG rats. TG(hA-1-7)L7301 rats also showed a greater capacity to withstand stress since TG rats showed a less pronounced deposition of collagen type III and fibronectin induced by isoproterenol treatment in the subendocardial area than in corresponding controls. In addition, hearts from TG rats showed reduced incidence and duration of reperfusion arrhythmias in comparison with SD rats. CONCLUSION: These results indicate that Ang-(1-7) has blood pressure-independent, antifibrotic effects, acting directly in the heart. less...

To elucidate the mechanism of cyclic nucleotides, such as adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5' -cyclic monophosphate (cGMP), in the regulation of human gastric motility, we examined the effects of forskolin (FSK), isoproterenol (ISO) and sodium nitroprusside (SNP) on the spontaneous, high K(+) and acetylcholine (ACh)-induced contractions of corporal circular smooth muscle in human stomach. Gastric circular smooth muscle showed regular spontaneous contraction, and FSK, ISO and SNP inhibited its phasic contraction and basal tone in a concentration-dependent manner. High K(+) (50 mM) produced sustained tonic contraction, and ACh (10 microM) produced initial transient contraction followed by later sustained tonic contraction with superimposed phasic contractions. FSK, ISO and SNP inhibited high K(+)-induced tonic contraction and also ACh-induced phasic and tonic contraction in a reversible manner. Nifedipine (1 microM), inhibitor of voltage-dependent L-type calcium current (VDCC(L)), almost abolished ACh-induced phasic contractions. These findings suggest that FSK, ISO and SNP, which are known cyclic nucleotide stimulators, inhibit smooth muscle contraction in human stomach partly via inhibition of VDCC(L). less...

Introduction- When sinus node or Av-node cells are damaged by disease, the implantation of an artificial cardiac pacemaker becomes necessary. In search for a biological alternative, the objective of this study was to demonstrate whether in-vivo adenoviral gene transfer of Adenylate-Cyclase type VI (ACVI) can create biological pacemaker activity in a porcine atrioventricular node (AV-node) block model. Genetic therapy of arrhythmic disorders of the heart has been subject of extensive studies. Cyclic AMP (cAMP) is generated in response to Beta-adrenergic receptor stimulation and also binds to HCN channels, where it regulates spontaneous rhythmic activity in the sinus node Materials and Methods- Adenoviruses encoding either AC VI or Beta-Galactosidase (lacZ) gene were injected into the lateral wall of the left ventricle of adult pigs via anterolateral thoracotomy at a dose of 1010 virus particles each. After 12 days, the AV-node was ablated and three-dimensional electrophysiological cardiac mapping was performed using the Ensite electro-anatomical system. Results- After rapid ventricular pacing and administration of Isoprenalin all animals of the AC-VI group exhibited an escape rhythm originating from the area of the left ventricular injection site at a rate of 100 + 7 beats/minute (n = 5), while the escape rhythms in the control group (n = 4) originated from the right ventricle. Western blot analysis of the injection sites revealed a significantly higher expression of AC-VI in the respective group as compared to the control group. Conclusions- Our study demonstrates that AC-VI gene transfer has the potential to create a biological pacemaker system. less...

Leptin is a circulating adipocyte-derived hormone that influences blood pressure (BP) and metabolism. This study was designed to define the possible role of leptin in regulation of the atrial natriuretic peptide (ANP) system using acute and chronic experiments. Intravenous infusion of rat leptin (250 mug/kg injection plus 2 mug*kg(-1)*min(-1) for 20 min) into Sprague-Dawley rats increased blood pressure (BP) by 25 mm Hg and decreased plasma level of ANP from 80.3 +/- 3.45 to 51.8 +/- 3.3 pg/ml. Reptinization attenuated the rise in BP but not the reduction of plasma ANP during leptin infusion. N-omega-l-arginine methyl ester (L-NAME) prevented the effects of leptin on the reduction of ANP level. In hyperleptinemic rats received adenovirus containing rat leptin cDNA (AdCMV-leptin), BP increased during first two days and then recovered to control value. Plasma concentration of ANP and expression of atrial ANP mRNA, but not of atrial ANP, in hyperleptinemic rats were lower than in the control groups on the first and second week after administration of AdCMV-leptin. These effects were not observed by the pretreatment with L-NAME. No differences in renal function and ANP receptor density in the kidney were found between hyperleptinemic and control rats. Basal ANP secretion and isoproterenol-induced suppression of ANP secretion from isolated perfused atria of hyperleptinemic rats were not different from those of other control groups. These data suggest that leptin inhibits ANP secretion indirectly through nitric oxide without changing basal or isoproterenol-induced ANP secretion. Key words: leptin, ANP, sympathetic nervous system, hypertension. less...