Rat Atria Research Articles

SEARCH FOR THE COMPOUNDS WHICH PROLONG EFFICIENT REFRACTORY PERIOD AMONG AMINOBENZOTHIAZOLES DERIVATIVES

The article reviews the experimental research in vitro on a revelation of effective refractory period prolongation properties in aminobenzothiazoles derivatives for isolated atrium of rats. We have provided the comparative characteristics of these compounds in relation with amiodarone.

Antiarrhythmic Mechanisms of SK Channel Inhibition in the Rat Atrium.

SK channels have functional importance in the cardiac atrium of many species, including humans. Pharmacological blockage of SK channels has been reported to be antiarrhythmic in animal models of atrial fibrillation; however, the exact antiarrhythmic mechanism of SK channel inhibition remains unclear. We speculated that together with a direct inhibition of repolarizing SK current, the previously observed depolarization of the atrial resting membrane potential (RMP) after SK channel inhibition reduces sodium channel availability, thereby prolonging the effective refractory period and slowing the conduction velocity (CV). We therefore aimed at elucidating these properties of SK channel inhibition and the underlying antiarrhythmic mechanisms using microelectrode action potential (AP) recordings and CV measurements in isolated rat atrium. Automated patch clamping and two-electrode voltage clamp were used to access INa and IK,ACh, respectively. The SK channel inhibitor N-(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (ICA) exhibited antiarrhythmic effects. ICA prevented electrically induced runs of atrial fibrillation in the isolated right atrium and induced atrial postrepolarization refractoriness and depolarized RMP. Moreover, ICA (1-10 μM) was found to slow CV; however, because of a marked prolongation of effective refractory period, the calculated wavelength was increased. Furthermore, at increased pacing frequencies, SK channel inhibition by ICA (10-30 μM) demonstrated prominent depression of other sodium channel-dependent parameters. ICA did not inhibit IK,ACh, but at concentrations above 10 μM, ICA use dependently inhibited INa. SK channel inhibition modulates multiple parameters of AP. It prolongs the AP duration and shifts the RMP towards more depolarized potentials through direct ISK block. This indirectly leads to sodium channel inhibition through accumulation of state dependently inactivated channels, which ultimately slows conduction and decreases excitability. However, a contribution from a direct sodium channel inhibition cannot be ruled. We here propose that the primary antiarrhythmic mechanism of SK channel inhibition is through direct potassium channel block and through indirect sodium channel inhibition.

Quantitative assay of brain natriuretic peptide in rat cardiomyocytes in the early postreperfusion period

We studied the intensities of accumulation and secretion of brain natriuretic peptide (BNP) in the early postreperfusion period, i.e., 5 min and 1 h after the start of reperfusion in the myocardium of the right atrium of rats. Total ischemia was modeled using Korpachev’s method. Immunocytochemical detection of peptide distribution in secretory cardiomyocytes was performed in ultrathin sections using polyclonal antibodies to BNP. The immunoreactive granules of A and B types were counted in optical fields of 38 × 38 µm2 using a transmission electron microscope. Five minutes after the start of reperfusion, the number of BNPcontaining secretory granules was similar to that observed in the control animals. After a 1-h postreperfusion period, we observed active formation and release of BNP because the number of granules of A and B-types increased by 134 and 210%, respectively, as compared to the intact group. These data demonstrate the stimulating effects of ischemic and reperfusion factors on the processes of synthesis and secretion of BNP in this period.

PP.28.09

Objective: Sustained beta-adrenoceptor activation is deleterious to the heart due to induction of pathological remodeling and compromising cardiomyocytes viability that increase a risk for sudden cardiac death. Our and others studies suggest that melatonin and omega-3 fatty acids are cardioprotective but underlying mechanisms are not fully elucidated. We hypothesize that both compounds may affect extracellular matrix remodeling by modulation of MMPase activity. We aimed to investigate MMP-2 activity in plasma, aorta and heart of beta-agonist isoproterenol (iso) injured normotensive (Wistar) and hypertensive rats (SHR) in respect to omega-3 fatty acids (Omacor) and melatonin treatments. Design and method: Wistar and SHR were divided into four groups: controls without treatment; iso-injured rats (118 mg/kg totally, s.c., for 7 days); iso-injured rats treated with melatonin (10 mg/kg, p.o., 2 month); iso-injured rats treated with Omacor (1.68 g/kg, p.o., 2 month). MMP-2 activity was estimated using gelatin zymography. Results: There was higher plasma but lower heart tissue-related MMP-2 activity in non-treated SHR vs Wistar rats and no difference in aorta. Iso caused an increase of MMP-2 in plasma as well as atria and decrease in left as well as right ventricles of Wistar rats. In contrast, iso decreased MMP-2 in plasma, aorta and increased in left and right ventricles of SHR. Melatonin and Omacor suppressed plasma MMP-2 activity in iso-treated Wistar but not iso-SHR. Both compounds decreased MMP-2 activity in atria of iso-Wistar rats but increased in iso-SHR as well as decreased MMP-2 activity in the ventricles of iso-treated SHR only. Conclusions: These data indicates that both melatonin and Omacor modulate circulating and cardiovascular tissue-related MMP-2 activity and there are strain- and tissue-related differences.

Characteristics of dendroaspis natriuretic peptide and its receptor in streptozotocin-induced diabetic rats.

Dendroaspis natriuretic peptide (DNP) shares a functionally important sequence homology with other natriuretic peptides. However, the characteristics of DNP and its receptor in the context of diabetes remafin to be fully elucidated. In the present study, alterations in the plasma levels and tissue contents of DNP and the properties of its receptor in diabetic rats, induced by streptozotocin (STZ) injection, were investigated. The plasma levels of DNP were 90.01 ± 4.12 and 196.68 ± 5.60 pg/ml in the control and STZ-induced diabetic rats, respectively. The tissue contents of DNP in the cardiac atrium, ventricle, renal cortex and inner medulla of the STZ-induced diabetic rats were also significantly increased compared with the control rats. Specific (125)I-DNP-binding sites were located predominantly in the glomeruli and inner medulla of the rat kidney. In the glomeruli of the kidney, the apparent dissociation constants (Kd) of (125)I-DNP in the control and STZ-induced diabetic rats were 0.41 ± 0.03 and 0.56 ± 0.06 nM, respectively. The maximum binding capacities (Bmax) of (125)I-DNP in control and STZ-induced diabetic rats were 2.98 ± 0.21 and 6.22 ± 1.06 fmol/mg protein, respectively. However, no differences were observed in the apparent Kd and Bmax of (125)I-DNP in the inner medulla of the kidney between the control and STZ-induced diabetic rats. In the glomerular and inner medullary kidney membranes, DNP stimulated the production of cyclic guanosine monophosphate (cGMP) in a dose-dependent manner. The magnitude of cGMP production in glomerular membranes was greater in the STZ-induced diabetic rats, whereas the magnitude of cGMP production in the inner medullary membranes was lower in the STZ-induced diabetic rats compared with the control rats. These results indicated that STZ-induced diabetes modulate DNP and its receptor, and also suggested that modulation of the DNP system is involved in the renal function of diabetic animals via the intracellular domain of the kidney NP receptor.

Improved blood–brain barrier distribution: Effect of borneol on the brain pharmacokinetics of kaempferol in rats by in vivo microdialysis sampling

Ethnopharmacological relevanceKaempferol (KA) exists in a variety of herbal medicines. In vitro and in vivo studies have focused on the anti-Alzheimer effect of KA. However, little is known about its brain pharmacokinetic profile. The accumulated amount of KA in brain is very low because of the protection of blood–brain barrier (BBB). Borneol (BO) is a classical aromatic refreshing traditional Chinese medicine and commonly used as an adjuvant component of traditional Chinese medicines (e.g. compound Danshen dropping pills) in the treatment of cardiovascular and cerebrovascular diseases. According to the basic theories of traditional Chinese medicine, BO is called an “upper guiding drug”, which can guide other components to the targeting tissues or organs in the upper part of the body, especially in the brain. Materials and methodsThe probes for blood and brain sampling were implanted within the jugular vein/right atrium and right hippocampus of SD rats, respectively. Rats were intravenous administered of KA (25mg/kg) alone or combined with BO (15, 30mg/kg) via caudal vein. The blood and brain microdialysates were collected every 15min for 180min and every 30min for 180−300min. A selective and sensitive high performance liquid chromatography–chemiluminescence method was developed for the determination of unbound KA in rat blood and brain microdialysates, which can be converted to their actual free-form concentrations based on the in vivo relative recoveries of KA across microdialysis probes. ResultsKA quickly crossed the BBB to enter the extracellular fluid of hippocampus and reached the maximum concentration of 0.11μg/mL within 30min. The brain bioavailability and brain delivery of KA evidently increased with the co-administration of 15 and 30mg/kg of BO. The AUC0–inf of KA in brain increased 1.84 and 2.19 times, and the Cmax of KA in brain increased 2.09 and 3.18 times than that without BO, respectively. In addition, the brain-to-blood distribution ratio of KA increased by 48.68% and 57.97% compared with that without BO. However, no significant difference in the T1/2 of unbound KA in blood aserved between three groups. ConclusionsBO can enhance the BBB permeability and improve the transportation of KA to brain. The dose-dependent effect of BO on the brain pharmacokinetic parameters of KA was observed. This co-administration strategy can be designed to enhance the brain accumulation of other neuropsychiatric medications.

Differential <i>β</i><sub>1</sub> Cardiac Adrenoceptor Modulation of Nitric Oxide Isoforms by <i>β</i><sub>1</sub> IgG from Patients with Periodontitis during Short-Term Hypoxia

Background: We demonstrated previously that serum IgG from periodontitis patients interacting with the second extracellular loop of the human cardiac β1 adrenoreceptors triggers the production of second messengers. In this paper we quantified the production of nitrates/nitrites and nitric oxide (NO), which in turn induces nitric oxide synthase (NOS) mRNA expression. Methods: We determined using β1 IgG, NOS activity isoforms, NOS expression, nitrate/nitrite assay, cGMP accumulation and PKC activity. Results: We established that serum β1 IgG autoantibodies and NO might be considered as early markers in normoxia/hypoxia system in rat isolated atria. The β1 IgG autoantibodies from periodontitis patients, while stimulating myocardial atria β1 adrenoreceptors, exert an increase on NO levels indirectly quantified as nitrite/nitrate, which acts as NO-storage molecules with significant increase in neuronal NOS (nNOS) and inducible NOS (iNOS) mRNA levels in hypoxia conditions. The significant increase in nNOS/iNOS mRNA and NOS activity as well as in NO levels after short-time hypoxia in rat isolated atria was detected. The expression of these genes are related with the increase in atria dF/dt, cyclic GMP (cGMP) and protein kinase C (PKC) activity and resemble the results obtained by Isoproterenol, an β1 adrenoreceptor agonist. Conclusion: These findings indicate that short-term hypoxia up-regulated rat atria NO/NOS system in the presence of β1 IgG autoantibodies shows that an antibody interacting with rat atria β1 adrenoreceptor can act as expression inducer of proinflammatory NO and its metabolites and that it might be useful and helps to maintain heart function and to prevent necrosis and subsequent loss of heart function during hypoxia.

Effects of 3-methyladenine on isolated left atria subjected to simulated ischaemia-reperfusion.

Although autophagy is a prominent feature of myocardial ischaemia and reperfusion, its functional significance is unclear and controversial. In order to gain a deeper insight into the role of autophagy in myocardial ischaemia-reperfusion, we explored the effects of the pharmacological inhibitor of autophagy 3-methyladenine (3-MA). Isolated rat atria subjected to simulated 75-min ischaemia/75-min reperfusion (Is-Rs) in the presence or absence of 3-MA were used. The LC3-II/LC3-I ratio, an indicator of autophagosome formation, did not increase after ischaemia either in the presence or absence of 3-MA, but there was significant enhancement during reperfusion, which was prevented by the presence of 3-MA. The autophagy inhibitor also increased p62 protein, one of the specific substrates degraded through the autophagy-lysosomal pathway. Electron micrographs showed double membrane autophagosome-like structures during reperfusion, which were absent in atria subjected to Is-Rs in the presence of 3-MA. These findings suggest that this agent inhibited the autophagic flux under the present experimental conditions. Inhibition of autophagy during Is-Rs was accompanied by a high incidence of tachyarrhythmias during reperfusion, and a decrease in the maximal inotropic response to β-adrenergic and to calcium stimulation at the end of Is-Rs. Deterioration of mitochondrial morphology and function, without affecting cell viability, was observed in atria subjected to Is-Rs in the presence of 3-MA. The present results suggest an association between the inhibition of autophagy and functional alterations of the cells that have undergone sublethal stress, and have been able to recover in this experimental model of ischaemia-reperfusion.

13Pulmonary hypertension leads to dysfunction and widespread ion channel remodelling within the atrioventricular node in the monocrotaline rat model

Introduction: Arrhythmias are common in pulmonary arterial hypertension (PAH) causing severe symptoms and sudden death. Atrioventricular (AV) node dysfunction is seen in PAH but the cause has not been determined. We assessed AV node function, and key ion channels, connexins and intracellular calcium handling proteins in the AV node in the moncrotaline (MCT) rat model of PAH. Methods: Male Wistar rats (200 g) were injected with either MCT (60 mg/kg) or saline (CON). Echo was performed to confirm PAH in the MCT rats. The rats were sacrificed if they showed clinical deterioration or on day 28. For whole heart experiments the heart was excised and mounted on a Langendorff perfusion column at 37°C. For right atrial (RA)/atrioventricular (AV) node experiments, a tissue preparation was superfused at 37°C. Atrio-His (AH) interval, AV effective refractory period (AVERP) and AV functional refractory period (AVFRP) were measured. A separate cohort of hearts was used to assess mRNA expression of key ion channels, connexins and intracellular calcium handling proteins in the AV node using reverse transcription quantitative polymerase chain reaction (RTqPCR). Sections of the AV node were labelled for HCN4 and connexin 43 using immunofluorescence allowing identification of the atrium, ventricle and four regions within the AV node: transitional tissue (TT), inferior nodal extension (INE), compact node (CN) and penetrating bundle (PB). Each region was microdissected and mRNA levels of 96 proteins were measured. Results: The whole heart experiments showed an increase in AVERP (87±1 ms in CON v 94±2 ms in MCT, p<0.05) and AVFRP (115±2 ms in CON v 122±2 ms in MCT, p<0.05) in the MCT rats compared with the CON rats. The isolated RA/AV node preparation demonstrated a 50% incidence of complete heart block in the MCT rats compared with 0% in the CON rats (p<0.05). The RTqPCR experiments demonstrated widespread remodelling of ion channel expression in the AV node in the MCT injected rats. In the TT, there was downregulation of phospholamban and sarcolipin; in the INE there was downregulation of HCN4, Kv2.1, minK and KChiP2, Kir2.4, phospholamban, RyR2, sarcolipin, α1 Na+/K+ ATPase and α2 Na+/K+ ATPase; in the CN there was downregulation of HCN4 and KChiP2. No significant changes were detected in the PB. Changes in HCN4 are shown. Conclusions: PAH leads to AV node dysfunction in the MCT model which is likely due to remodelling of ion channels and intracellular calcium handling proteins in the AV node. Downregulation of HCN4 is of particular interest given recent studies showing that HCN4 knockout causes heart block and suggests HCN4 may play a greater role in AV node function than has previously been realised. ![Graphic][1] Figure HCN4 expression in the AV node. The control rats show the expected distribution of HCN4 across the different regions of the AV node. Significant downregulation of HCN4 is seen in the atrium, INE and CN of the MCT rats [1]: /embed/inline-graphic-1.gif

The effect of the left stellate ganglion on sympathetic neural remodeling of the left atrium in rats following myocardial infarction.

The neural remodeling of the atrium plays an important role in the initiation of atrial fibrillation after myocardial infarction (MI); however, the effects of the left stellate ganglion (LSG) on the neural remodeling of the atrium remain incompletely understood. Thus, this study investigated the mechanism by which the LSG mediates sympathetic neural remodeling of the left atrium (LA) in rats after MI. Sixty rats were randomly divided into a Sham group and an MI group. The expression levels of growth-associated protein-43 (GAP43) and nerve growth factor (NGF) messenger ribonucleic acid (mRNA) were measured by reverse transcription polymerase chain reaction. Immunohistochemistry was used to detect the distribution and density of GAP43- and NGF-positive nerves. The expression levels of the proteins were quantified by Western blotting. Compared with the Sham group, GAP43 mRNA expression in the LSG was increased in the MI group (P < 0.01), but not significantly increased in the LA. Immunohistochemical analysis demonstrated that in both the LSG and the LA, the mean densities of GAP43- and NGF-positive nerves in the MI group were increased (P < 0.01). In both the LSG and the LA, the protein levels of GAP43 and NGF in the MI group were increased relative to the Sham group (P < 0.01). The increased levels of NGF and GAP43 proteins can induce sympathetic nerve hyperinnervation in the LSG and the LA after MI. The increased GAP43 proteins in the LA, which may have been transported from the LSG, accelerated LA sympathetic neural remodeling in rats.

The effect of the Ras homolog gene family (Rho), member A/Rho associated coiled-coil forming protein kinase pathway in atrial fibrosis of type 2 diabetes in rats.

Diabetes mellitus promotes atrial structural remodeling, thereby producing atrial arrhythmogenicity. Atrial arrhythmia can substantially increase the risk of premature death. The aim of this study was to investigate the role of Ras homolog gene family, member A (RhoA)/Rho associated coiled-coil forming protein kinase (ROCK) in atrial fibrosis in diabetic hearts, and the effects of fasudil hydrochloride hydrate on atrial fibrosis. An eight-week-old male Sprague-Dawley rat model of type 2 diabetes was established using a high-fat diet combined with streptozotocin [30 mg/kg, once, intraperitoneal (i.p.)]. Animals were randomly divided into three groups: Control rats, untreated diabetic rats that received vehicle, and treated diabetic rats that received Rho kinase inhibitor fasudil hydrochloride hydrate (10 mg/kg/day, i.p., for 14 weeks). The morphological features of atrial fibrosis were observed using Masson staining. The mRNA expression levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen were assessed with quantitative polymerase chain reaction. The protein levels of RhoA, ROCK1 and ROCK2 were evaluated using western blot analysis. The atria of untreated diabetic rats showed evident atrial fibrosis as compared to the control rats; the mRNA expression levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen were upregulated; and the protein levels of RhoA, ROCK1 and ROCK2 were increased. The treatment with fasudil hydrochloride hydrate significantly reduced atrial fibrosis, mRNA levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen, and the protein levels of RhoA, ROCK1 and ROCK2. The results suggested that RhoA/ROCK was involved in atrial fibrosis, and that fasudil hydrochloride hydrate ameliorates atrial fibrosis through the RhoA/ROCK pathway in rats with type 2 diabetes.

P109Opposite actions of atrial adenosine A1 receptor on G protein-coupled inwardly-rectifying potassium (GIRK/Kir3.1/3.4) and small Ca2+-activated K+ outward (KCa2/SK) currents

Purpose: Adenosine fine-tuning regulates cardiovascular function. The negative chronotropic effect of adenosine depends on the activation of the most abundant adenosine receptor in the heart, the A1AR, leading to GIRK/Kir3.1/3.4 channels opening in the SA node. The A1AR-mediated responses in the atrial muscle also depend on KCa2/SK channel function (unpublished observations). Since these channels are involved in atrial repolarization and in human rhythm disturbances, we aimed at studying the interplay between A1AR and SK channels both in situ and single-cell preparations of rat atria. Methods: Isometric contraction experiments were performed on isolated spontaneously beating atria from Wistar rats. For whole-cell voltage-clamp recordings, atrial myocytes were enzymatically isolated by retrograde perfusion. Immunolocalization studies (A1AR, GIRK1, KCa2.2 and KCa2.3) were evaluated by confocal microscopy. The SA node was identified as a connexin-43 low / neurofilament-160 high immunoreactive region. Results: The selective A1AR agonist R-PIA (0.001-1μM) decreased atrial chronotropism and inotropism in concentration-dependent manner. However, the negative chronotropic effect of R-PIA (IC50~0.03 μM) was evidenced at much lower (~30 times) concentrations than the negative inotropic action (IC50~1 μM). Selective blockade of A1AR and of GIRK/Kir3.1/3.4 channels, respectively with DPCPX (100 nM) and tertiapin Q (300 nM), significantly attenuated the effects R-PIA. Blockade of KCa2/SK channels with apamin (30 nM) potentiated the negative inotropic response of R-PIA, without affecting atrial rate. Apamin-induced atrial sensitization to R-PIA was not observed with other cardiodepressant agents, namely oxotremorine (0.01-3 μM). Voltage-clamp experiments demonstrated that adenosine, via A1AR, plays a dual role in atrial cardiomyocytes by activating inwardly rectifying GIRK/Kir3.1/3.4 and inactivating outward Ca2+-activated KCa2/SK currents upon cell depolarization. Immunolabelling of A1AR, KIR3.1, KCa2.2 and KCa2.3 was observed throughout the right atria. Conclusion: Data demonstrate for the first time that A1AR has opposing effects on distinct K+ currents operating atrial repolarization. While A1AR activation favors the opening of inwardly rectifying GIRK/KIR3.1/3.4 channels modulating atrial automatism, it may also inactivate outward KCa2/SK currents. Taking into consideration that KCa2/SK channel dysfunction has pro-arrhythmic effects drugs targeting the A1AR for supraventricular tachycardia must be used with caution as they might precipitate atrial arrhythmias.

Variable t-tubule organization and Ca2+ homeostasis across the atria.

Although t-tubules have traditionally been thought to be absent in atrial cardiomyocytes, recent studies have suggested that t-tubules exist in the atria of large mammals. However, it is unclear whether regional differences in t-tubule organization exist that define cardiomyocyte function across the atria. We sought to investigate regional t-tubule density in pig and rat atria and the consequences for cardiomyocyte Ca(2+) homeostasis. We observed t-tubules in approximately one-third of rat atrial cardiomyocytes, in both tissue cryosections and isolated cardiomyocytes. In a minority (≈10%) of atrial cardiomyocytes, the t-tubular network was well organized, with a transverse structure resembling that of ventricular cardiomyocytes. In both rat and pig atrial tissue, we observed higher t-tubule density in the epicardium than in the endocardium. Consistent with high variability in the distribution of t-tubules and Ca(2+) channels among cells, L-type Ca(2+) current amplitude was also highly variable and steeply dependent on capacitance and t-tubule density. Accordingly, Ca(2+) transients showed great variability in Ca(2+) release synchrony. Simultaneous imaging of the cell membrane and Ca(2+) transients confirmed t-tubule functionality. Results from mathematical modeling indicated that a transmural gradient in t-tubule organization and Ca(2+) release kinetics supports synchronization of contraction across the atrial wall and may underlie transmural differences in the refractory period. In conclusion, our results indicate that t-tubule density is highly variable across the atria. We propose that higher t-tubule density in cells localized in the epicardium may promote synchronization of contraction across the atrial wall.

Calcium influx inhibition is involved in the hypotensive and vasorelaxant effects induced by yangambin.

The pharmacological effects on the cardiovascular system of yangambin, a lignan isolated from Ocotea duckei Vattimo (Lauraceae), were studied in rats using combined functional and biochemical approaches. In non-anaesthetized rats, yangambin (1, 5, 10, 20, 30 mg/kg, i.v.) induced hypotension (−3.5 ± 0.2; −7.1 ± 0.8; −8.9 ± 1.3; −14 ± 2.3, −25.5% ± 2.6%, respectively) accompanied by tachycardia (5.9 ± 0.5; 5.9 ± 1.6; 8.8 ± 1.4; 11.6, 18.8% ± 3.4%, respectively). In isolated rat atria, yangambin (0.1 µM–1 mM) had very slight negative inotropic (Emax = 35.6% ± 6.4%) and chronotropic effects (Emax = 10.2% ± 2.9%). In endothelium-intact rat mesenteric artery, yangambin (0.1 µM–1 mM) induced concentration-dependent relaxation (pD2 = 4.5 ± 0.06) of contractions induced by phenylephrine and this effect was not affected by removal of the endothelium. Interestingly, like nifedipine, the relaxant effect induced by yangambin was more potent on the contractile response induced by KCl 80 mM (pD2 = 4.8 ± 0.05) when compared to that induced by phenylephrine. Furthermore, yangambin inhibited CaCl2-induced contractions in a concentration-dependent manner. This lignan also induced relaxation (pD2 = 4.0 ± 0.04) of isolated arteries pre-contracted with S(−)-Bay K 8644. In fura-2/AM-loaded myocytes of rat mesenteric arteries, yangambin inhibited the Ca2+ signal evoked by KCl 60 mM. In conclusion, these results suggest that the hypotensive effect of yangambin is probably due to a peripheral vasodilatation that involves, at least, the inhibition the Ca2+ influx through voltage-gated Ca2+ channels.

Effects of eugenol on resting tension of rat atria

In cardiac and skeletal muscle, eugenol (μM range) blocks excitation-contraction coupling. In skeletal muscle, however, larger doses of eugenol (mM range) induce calcium release from the sarcoplasmic reticulum. The effects of eugenol are therefore dependent on its concentration. In this study, we evaluated the effects of eugenol on the contractility of isolated, quiescent atrial trabeculae from male Wistar rats (250-300 g; n=131) and measured atrial ATP content. Eugenol (1, 3, 5, 7, and 10 mM) increased resting tension in a dose-dependent manner. Ryanodine [100 µM; a specific ryanodine receptor (RyR) blocker] and procaine (30 mM; a nonspecific RyR blocker) did not block the increased resting tension induced by eugenol regardless of whether extracellular calcium was present. The myosin-specific inhibitor 2,3-butanedione monoxime (BDM), however, reversed the increase in resting tension induced by eugenol. In Triton-skinned atrial trabeculae, in which all membranes were solubilized, eugenol did not change resting tension, maximum force produced, or the force vs pCa relationship (pCa=-log [Ca2+]). Given that eugenol reduced ATP concentration, the increase in resting tension observed in this study may have resulted from cooperative activation of cardiac thin filaments by strongly attached cross-bridges (rigor state).

Human epicardial adipose tissue induces fibrosis of the atrial myocardium through the secretion of adipo-fibrokines

Recent studies have reported a relationship between the abundance of epicardial adipose tissue (EAT) and the risk of cardiovascular diseases including atrial fibrillation (AF). However, the underlying mechanisms are unknown. The aim of this study was to examine the effects of the secretome of human EAT on the histological properties of the myocardium. Samples of EAT and subcutaneous adipose (SAT), obtained from 39 patients undergoing coronary bypass surgery, were analysed and tested in an organo-culture model of rat atria to evaluate the fibrotic properties of human fat depots. The EAT secretome induced global fibrosis (interstitial and peripheral) of rat atria in organo-culture conditions. Activin A was highly expressed in EAT compared with SAT and promoted atrial fibrosis, an effect blocked using neutralizing antibody. In addition, Activin A levels were enhanced in patients with low left-ventricular function. In sections of human atrial and ventricular myocardium, adipose and myocardial tissues were in close contact, together with fibrosis. This study provides the first evidence that the secretome from EAT promotes myocardial fibrosis through the secretion of adipo-fibrokines such as Activin A.

Regulation of KV1.5 Channel Density in the Rat Atria

The density of functional Kv1.5 channels underlying IKur, the main repolarizing current in human atria, is a result of the equilibrium between exocytosis and endocytosis. We have shown that in addition to constitutive exocytosis, Kv1.5 channels can also undergo regulated exocytosis, for instance following changes in the mechanical environment or changes in the cholesterol content of the sarcolemma. Although constitutive and triggered endocytosis have been investigated for Kv1.5, its internalization pathway has not been described. Using high-resolution 3-D deconvolution microscopy, we showed that Kv1.5 channels are associated with clathrin vesicles (CVs) in atrial myocytes. Electron microscopy (EM) showed that CVs are found both at the intercalated disc and at the lateral sarcolemma, aligned along z-bands. Blockade of the clathrin pathway using hypertonic media or SiRNA increased IKur densityin atrial myocytes and led to Kv1.5 channels accumulating at the sarcolemma, as shown by biotinylation assays and fluorescence recovery after photobleaching (FRAP) experiments. These data support the hypothesis that Kv1.5 channels are internalized via the clathrin pathway.Next, we investigated Kv1.5 channel internalization in a rat model of atrial hemodynamic overload. Despite reduced Kv1.5 protein expression in dilated atria, IKur density was unchanged, suggesting increased functional Kv1.5 channels at the sarcolemma. Clathrin expression was reduced in dilated atria, and a decreased colocalization between Kv1.5 channels and CVs was observed. However, EM showed no significant difference in internalization activity between sham and dilated atria. Therefore, the reduced clathrin protein synthesis observed in dilated atria is not likely to be responsible for the accumulation of Kv1.5 channels at the sarcolemma. Other mechanisms such as increased recycling and/or membrane stabilization must be investigated to understand how IKur is maintained in dilated atria.

PGE&lt;sub&gt;2&lt;/sub&gt; Generation in Myocardium from Isolated Rat Atrium under Hypoxia and Reoxygenation Conditions. Effect of Anti-&lt;i&gt;β&lt;/i&gt;&lt;sub&gt;1&lt;/sub&gt; IgG from Patients with Chronic Severe Periodontitis

Background: Hypoxia is one of the most frequently encountered stresses in health and disease. Methods: We compared the effects of an anti-β1 periodontal IgG (pIgG) and an authentic β1 adrenergic agonist, xamoterol, on isolated myocardium from rat atria contractility. We used an ELISA assay to measure the generation of PGE2 in vitro after the addition of either the antibody or the adrenergic agonist. We analyzed the myocardium histopathologically in the presence of both the antibody and/or the adrenergic agonist drug during normoxia, hypoxia and reperfusion conditions. Results: PGE2 generation increased during the hypoxia and was unchanged during reoxygenation period compared with the production of this prostanoid in atria during normoxia condition. A β1 specific adrenoceptor antagonist atenolol and the β1 synthetic peptide abrogated the increment of the prostanoid in the presence of pIgG but only atenolol due to it in the presence of xamoterol. The increment of PGE2 was dependent on the activation of cox-1 and cox-2 isoforms. Moreover, cox-2 was more active and produced more increments in the production of PGE2 in the presence of the pIgG than cox-1 activation. Histopathologically, studies of myocardium specimens during these different periods of the experimental protocol: basal (B), hypoxia (H) and reoxygenation (R), were also performed and showed tissue necrosis and edematization at the myocardium level. Conclusion: The phenomenon studied here supports the notion that PGE2 may be responsible for tissue edematization. PGE2 maybe acts as a beneficial modulator in the myocardium and prevents a major injury of it. The inflammation damage to the heart organ and cardiomyocytes caused by the actions of the antibodies in the course of heart lesions provoked by cardiovascular autoimmune disease, explains some of these results obtained in the present experiments. Further studies will be needed to establish the real role of PGE2 during hypoxia injury of the heart in the course of autoimmune diseases.

Pacing-induced non-uniform ca(2+) dynamics in rat atria revealed by rapid-scanning confocal microscopy.

Intracellular Ca2+ ([Ca2+]i) dynamics in isolated myocytes differ between the atria and ventricles due to the distinct t-tubular distributions. Although cellular aspects of ventricular [Ca2+]i dynamics in the heart have been extensively studied, little is known about those of atrial myocytes in situ. Here we visualized precise [Ca2+]i dynamics of atrial myocytes in Langendorff-perfused rat hearts by rapid-scanning confocal microscopy. Of 16 fluo-4-loaded hearts imaged during pacing up to 4-Hz, five hearts showed spatially uniform Ca2+ transients on systole among individual cells, whereas no discernible [Ca2+]i elevation developed during diastole. In contrast, the remaining hearts showed non-uniform [Ca2+]i dynamics within and among the cells especially under high-frequency (4 Hz) excitation, where subcellular cluster-like [Ca2+]i rises or wave-like [Ca2+]i propagation occurred on excitation. Such [Ca2+]i inhomogeneity was more pronounced at high-frequency pacing, showing beat-to-beat Ca2+ transient alternans. Despite such non-uniform dynamics, cessation of burst pacing of the atria was not followed by emergence of spontaneous Ca2+ waves, indicating minor Ca2+-releasing potentials of the sarcoplasmic reticulum (SR). In summary, rat atria display a propensity to show non-uniform [Ca2+]i dynamics on systole due to impaired Ca2+-release from the SR and paucity of t-tubules. Our results provide an important basis for understanding atrial pathophysiology.

The Role of α7 Nicotinic Acetylcholine Receptor in Modulation of Heart Rate Dynamics in Endotoxemic Rats

Previous reports have indicated that artificial stimulation of the vagus nerve reduces systemic inflammation in experimental models of sepsis. This phenomenon is a part of a broader cholinergic anti-inflammatory pathway which activates the vagus nerve to modulate inflammation through activation of alpha7 nicotinic acetylcholine receptors (α7nACHR). Heart rate variability represents the complex interplay between autonomic nervous system and cardiac pacemaker cells. Reduced heart rate variability and increased cardiac cycle regularity is a hallmark of clinical conditions that are associated with systemic inflammation (e.g. endotoxemia and sepsis). The present study was aimed to assess the role of α7nACHR in modulation of heart rate dynamics during systemic inflammation. Systemic inflammation was induced by injection of endotoxin (lipopolysaccharide) in rats. Electrocardiogram and body temperature were recorded in conscious animals using a telemetric system. Linear and non-linear indices of heart rate variability (e.g. sample entropy and fractal-like temporal structure) were assessed. RT-PCR and immunohistochemistry studies showed that α7nACHR is expressed in rat atrium and is mainly localized at the endothelial layer. Systemic administration of an α7nACHR antagonist (methyllycaconitine) did not show a significant effect on body temperature or heart rate dynamics in naïve rats. However, α7nACHR blockade could further reduce heart rate variability and elicit a febrile response in endotoxemic rats. Pre-treatment of endotoxemic animals with an α7nACHR agonist (PHA-543613) was unable to modulate heart rate dynamics in endotoxemic rats but could prevent the effect of endotoxin on body temperature within 24 h experiment. Neither methyllycaconitine nor PHA-543613 could affect cardiac beating variability of isolated perfused hearts taken from control or endotoxemic rats. Based on our observations we suggest a tonic role for nicotinic acetylcholine receptors in modulation of heart rate dynamics during systemic inflammation.