Node Preparations Research Articles

Crowd-sourced AI based Indoor Localization using Support Vector Regression and Pedestrian Dead Reckoning

Aims and Background: Artificial intelligence (AI) is expanding in the market daily to assist humans in a variety of ways. However, as these models are expensive, there is still a gap in the availability of AI products to the common public with high component dependency. Objective and Methodology: To address the issue of additional component dependency on AI products, we propose a model that can use available Smartphone resources to perceive real-world huddles and assist ordinary people with their daily needs. The proposed AI model is to predict the user’s indoor position (Node) at the computer science and engineering block of CMR Institute of Technology (CMRIT) by using Smartphone sensors and wireless signals. We used SVR to predict the regular walk steps needed between two Nodes and Pedestrian Dead Reckoning (PDR) to predict the walk steps needed while the signal was lost in the indoor environment. Results: The Support vector regression (SVR) models make the locations to be available within the specified building boundaries for proper guidance. The PDR approach supports the user while signal loss between two Received Signal Strength Indicators (RSSI). The Pedestrian dead reckoning - Support Vector Regression (PD-SVR) results are showing 98% accuracy in NODE predictions with routing tables. The indoor positioning is 100% accurate with dynamic crowd-sourcing Node preparation. Conclusion: The results are compared with other indoor navigation models K-nearest neighbor (KNN) and DF-SVM are given 95% accurate NODE estimation with minimal need for network components.

Role of Endothelin-1 in Right Atrial Arrhythmogenesis in Rabbits with Monocrotaline-Induced Pulmonary Arterial Hypertension.

Atrial arrhythmias are considered prominent phenomena in pulmonary arterial hypertension (PAH) resulting from atrial electrical and structural remodeling. Endothelin (ET)-1 levels correlate with PAH severity and are associated with atrial remodeling and arrhythmia. In this study, hemodynamic measurement, western blot analysis, and histopathology were performed in the control and monocrotaline (MCT, 60 mg/kg)-induced PAH rabbits. Conventional microelectrodes were used to simultaneously record the electrical activity in the isolated sinoatrial node (SAN) and right atrium (RA) tissue preparations before and after ET-1 (10 nM) or BQ-485 (an ET-A receptor antagonist, 100 nM) perfusion. MCT-treated rabbits showed an increased relative wall thickness in the pulmonary arterioles, mean cell width, cross-sectional area of RV myocytes, and higher right ventricular systolic pressure, which were deemed to have PAH. Compared to the control, the spontaneous beating rate of SAN–RA preparations was faster in the MCT-induced PAH group, which can be slowed down by ET-1. MCT-induced PAH rabbits had a higher incidence of sinoatrial conduction blocks, and ET-1 can induce atrial premature beats or short runs of intra-atrial reentrant tachycardia. BQ 485 administration can mitigate ET-1-induced RA arrhythmogenesis in MCT-induced PAH. The RA specimens from MCT-induced PAH rabbits had a smaller connexin 43 and larger ROCK1 and phosphorylated Akt than the control, and similar PKG and Akt to the control. In conclusion, ET-1 acts as a trigger factor to interact with the arrhythmogenic substrate to initiate and maintain atrial arrhythmias in PAH. ET-1/ET-A receptor/ROCK signaling may be a target for therapeutic interventions to treat PAH-induced atrial arrhythmias.

Methylene Blue Near-Infrared Fluorescence Imaging in Breast Cancer Sentinel Node Biopsy.

Simple SummaryCurrently the gold standard for sentinel node biopsy in breast cancer patients is radioactive nanocolloid and a blue dye. In the age of fluorescence guided surgery new fluorophores are used and methylene blue presents some fluorescent properties. This study is the first in a clinical series presenting the possible use of methylene blue as a fluorescent dye for the identification of sentinel nodes in breast cancer sentinel node biopsy. We presented a feasibility of this new method and also in additional experiments because of the quenching effect limitation, found possible dilution of methylene blue presenting improved fluorescence. Introduction: Fluorescence-based navigation for breast cancer sentinel node biopsy is a novel method that uses indocyanine green as a fluorophore. However, methylene blue (MB) also has some fluorescent properties. This study is the first in a clinical series presenting the possible use of MB as a fluorescent dye for the identification of sentinel nodes in breast sentinel node biopsy. Material and methods: Forty-nine patients with breast cancer who underwent sentinel node biopsy procedures were enrolled in the study. All patients underwent standard simultaneous injection of nanocolloid and MB. We visualized and assessed the sentinel nodes and the lymphatic channels transcutaneously, with and without fluorescence, and calculated the signal-to-background ratio (SBR). We also analyzed the corresponding fluorescence intensity of various dilutions of MB. Results: In twenty-three patients (46.9%), the location of the sentinel node, or the end of the lymphatic path, was visible transcutaneously. The median SBR for transcutaneous sentinel node location was 1.69 (range 1.66–4.35). Lymphatic channels were visible under fluorescence in 14 patients (28.6%) prior to visualization by the naked eye, with an average SBR of 2.01 (range 1.14–5.6). The sentinel node was visible under fluorescence in 25 patients (51%). The median SBR for sentinel node visualization with MB fluorescence was 2.54 (range 1.34–6.86). Sentinel nodes were visualized faster under fluorescence during sentinel node preparation. Factors associated with the rate of visualization included diabetes (p = 0.001), neoadjuvant chemotherapy (p = 0.003), and multifocality (p = 0.004). The best fluorescence was obtained using 40 μM (0.0128 mg/mL) MB, but we also observed a clinically relevant dilution range between 20 μM (0.0064 mg/mL) and 100 μM (0.032 mg/mL). Conclusions: For the first time, we propose the clinical usage of MB as a fluorophore for fluorescence-guided sentinel node biopsy in breast cancer patients. The quenching effect of the dye may be the reason for its poor detection rate. Our analysis of different concentrations of MB suggests a need for a detailed clinical analysis to highlight the practical usefulness of the dye.

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk.

KCNMA1 encodes the α-subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. Our data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that KCa1.1 loss of function may predispose to AF.

A circadian clock in the sinus node mediates day-night rhythms in Hcn4 and heart rate

BackgroundHeart rate follows a diurnal variation, and slow heart rhythms occur primarily at night.ObjectiveThe lower heart rate during sleep is assumed to be neural in origin, but here we tested whether a day-night difference in intrinsic pacemaking is involved.MethodsIn vivo and in vitro electrocardiographic recordings, vagotomy, transgenics, quantitative polymerase chain reaction, Western blotting, immunohistochemistry, patch clamp, reporter bioluminescence recordings, and chromatin immunoprecipitation were used.ResultsThe day-night difference in the average heart rate of mice was independent of fluctuations in average locomotor activity and persisted under pharmacological, surgical, and transgenic interruption of autonomic input to the heart. Spontaneous beating rate of isolated (ie, denervated) sinus node (SN) preparations exhibited a day-night rhythm concomitant with rhythmic messenger RNA expression of ion channels including hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4). In vitro studies demonstrated 24-hour rhythms in the human HCN4 promoter and the corresponding funny current. The day-night heart rate difference in mice was abolished by HCN block, both in vivo and in the isolated SN. Rhythmic expression of canonical circadian clock transcription factors, for example, Brain and muscle ARNT-Like 1 (BMAL1) and Cryptochrome (CRY) was identified in the SN and disruption of the local clock (by cardiomyocyte-specific knockout of Bmal1) abolished the day-night difference in Hcn4 and intrinsic heart rate. Chromatin immunoprecipitation revealed specific BMAL1 binding sites on Hcn4, linking the local clock with intrinsic rate control.ConclusionThe circadian variation in heart rate involves SN local clock–dependent Hcn4 rhythmicity. Data reveal a novel regulator of heart rate and mechanistic insight into bradycardia during sleep.

Characterization of Magnesium Sulfate as an Antiarrhythmic Agent.

BACKGROUND: Recently, intravenous magnesium therapy has been used for the treatment of ventricular arrhythmias, but data to establish a causal link between the electrophysiological properties and the antiarrhythmic actions are lacking. METHODS AND RESULTS: The acute antiarrhythmic effect of magnesium sulfate was assessed using epinephrine-, digitalis-, and coronary ligation-induced canine ventricular arrhythmia models. The intravenous administration of magnesium sulfate (100 mg/kg) reduced the incidence of the ventricular arrhythmias of all models. The antiarrhythmic effect on the epinephrine-induced arrhythmia was potent and long-lasting, while those on the other arrhythmia models were weak and transient. The direct cardiovascular effects were assessed using the canine isolated, blood-perfused sinus node, papillary muscle, and atrioventricular node preparations. The intracoronary administration of magnesium sulfate (0.1-30 mg) suppressed sinoatrial automaticity and ventricular contraction, while it increased atrio-His and His-ventricular conduction time, coronary blood flow, and the duration of monophasic action potential in a dose-dependent manner. The effects on His-ventricular conduction and monophasic action potential duration were less potent compared with the other cardiovascular effects. CONCLUSIONS: These results suggest that magnesium sulfate possesses multiple electrophysiological properties and that the effects related to the calcium channel inhibition may be the most relevant for the antiarrhythmic actions.

Technical details for harvesting gastro-epiploic lymph node flaps via mini-laparotomy incision

Laparoscopic harvest method to obtain gastro-epiploic lymph node flaps for lymphedema treatment has been previously described. In this article, the technical details of an alternative method - open approach via mini-laparotomy incision - for harvesting gastro-epiploic lymph node flaps and preparation of the flaps for the inset is presented. A total of 17 patients were included in this series of the mini-laparotomy approach. Blood loss was minimal during the surgery. The average duration of lymph node flap harvest was 65min. The average length of hospital stay was 10 days. The period of the restricted diet was 1.5 days. The upper abdominal scar was acceptable, there were no postoperative hernia or bulging, and there were no complications related to bowel obstruction during the follow-up. The open approach harvest method via mini-laparotomy incision offers similar results to laparoscopic harvest method, and it is safe when applied with the right technique.

Atrial arrhythmogenesis in a rabbit model of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) increases the risk of atrial fibrillation (AF), however, its arrhythmogenic mechanisms are unclear. This study investigated the effects of COPD on AF triggers (pulmonary veins, PVs) and substrates (atria), and their potential underlying mechanisms. Electrocardiographic, echocardiographic, and biochemical studies were conducted in control rabbits and rabbits with human leukocyte elastase (0.3 unit/kg)-induced COPD. Conventional microelectrode, Western blotting, and histological examinations were performed on PV, left atrium (LA), right atrium, and sinoatrial node (SAN) preparations from control rabbits and those with COPD. The rabbits with COPD had a higher incidence of atrial premature complexes, PV burst firing and delayed afterdepolarizations, higher sympathetic activity, larger LA, and faster PV spontaneous activity than did the control rabbits; but they exhibited a slower SAN beating rate. The LA of the rabbits with COPD had a shorter action potential duration and longer tachyarrhythmia induced by tachypacing (20 Hz) and isoproterenol (1 μM). Additionally, the rabbits with COPD had higher fibrosis in the PVs, LA, and SAN. H89 (10 μM), KN93 (1 μM), and KB-R7943 (10 μM) significantly suppressed burst firing and delayed afterdepolarizations in the PVs of the rabbits with COPD. Moreover, compared with the control rabbits, those with COPD had lower expression levels of the β1 adrenergic receptor, Cav 1.2, and Na+/Ca2+ exchanger in the PVs; Cav 1.2 in the LA; and hyperpolarization-activated cyclic nucleotide-gated K+ channel 4 in the SAN. COPD increases atrial arrhythmogenesis by modulating the distinctive electrophysiological characteristics of the PVs, LA, and SAN.

Electrical Conduction System Remodeling in Streptozotocin-Induced Diabetes Mellitus Rat Heart.

Cardiovascular complications are common in type 1 diabetes mellitus (TIDM) and there is an increased risk of arrhythmias as a result of dysfunction of the cardiac conduction system (CCS). We have previously shown that, in vivo, there is a decrease in the heart rate and prolongation of the QRS complex in streptozotocin-induced type 1 diabetic rats indicating dysfunction of the CCS. The aim of this study was to investigate the function of the ex vivo CCS and key proteins that are involved in pacemaker mechanisms in TIDM. RR interval, PR interval and QRS complex duration were significantly increased in diabetic rats. The beating rate of the isolated sinoatrial node (SAN) preparation was significantly decreased in diabetic rats. The funny current density and cell capacitance were significantly decreased in diabetic nodal cells. Western blot showed that proteins involved in the function of the CCS were significantly decreased in diabetic rats, namely: HCN4, Cav1.3, Cav3.1, Cx45, and NCX1 in the SAN; RyR2 and NCX1 in the atrioventricular junction and Cx40, Cx43, Cx45, and RyR2 in the Purkinje network. We conclude that there are complex functional and cellular changes in the CCS in TIDM. The changes in the proteins involved in the function of this electrical system are expected to adversely affect action potential generation and propagation, and these changes are likely to be arrhythmogenic.

Pacemaking in cardiac tissue. From IK2 to a coupled-clock system.

Initially, diastolic depolarization in Purkinje fibers was explained by deactivation of gK2 in the presence of inward current. Weakness of the hypothesis was a too negative reversal potential, sensitivity to external Na+ ions, existence of K+ depletion, and fake current during hyperpolarizing clamps. The development of a sinus node preparation of almost microscopic dimensions allowing uniform voltage clamps created new possibilities. Three different groups discovered in this improved node preparation an hyperpolarization induced time‐dependent inward current, with a reversal potential positive to the resting potential, carried by a mixture of Na+ and K+ ions. A new current, If, or funny current was born. It is not the only pacemaker current. The following sequence of currents (membrane clock) has been proposed: diastole starts as a consequence of IK deactivation and If activation; followed by activation of the T‐type Ca2+ current, Ca2+‐induced Ca2+ release from the SR, and activation of sodium‐calcium exchange current with further depolarization of the membrane till threshold of the L‐type Ca2+current is reached. The release of Ca2+ can also occur spontaneously independently from a T‐type Ca2+current. The system acts then as a primary intracellular clock. The review is completed by description of an evolution in the direction of biological pacing using induced pluripotent stem cells or transcription factors. See also: https://doi.org/10.14814/phy2.13860 & https://doi.org/10.14814/phy2.13861

Abstract 16851: Sinus Node Dysfunction Underlies Chronotropic Incompetence in Rats With Heart Failure and Preserved Ejection Fraction

Introduction: Chronotropic incompetence is a major limiting factor of exercise capacity in patients with heart failure and preserved ejection fraction (HFpEF) but little is known about the mechanisms. Hypothesis: We tested the hypothesis that sinus node dysfunction underlies chronotropic incompetence in rats with HFpEF. Methods: Dahl salt-sensitive rats were fed high-salt diet (8% NaCl) to induce HFpEF. Rats fed normal-salt diet (0.3% NaCl) served as controls. Echocardiography was used to confirm the development of diastolic dysfunction in high-salt-fed rats. Telemetry devices were implanted to echo-verified HFpEF rats and treadmill exercise test was performed to assess heart rate response to exercise and exercise capacity. Sinus node recovery time (SNRT) was measured in vivo and ex vivo optical mapping was performed in isolated sinus node tissues to measure heart rate response to isoproterenol. Reverse transcriptase PCR was performed in RNA samples isolated from sinus node tissues. Results: HFpEF rats showed decreased E/A ratio (1.30±0.07 vs. 1.58±0.13, p<0.05) and increased E/E’ ratio (19.17±2.63 vs. 13.78±2.58, p<0.05) compared to controls, indicative of diastolic dysfunction. Ejection fractions were normal (71±7% vs. 73±3%, p=ns) in both groups. Resting blood pressure were elevated in HFpEF rats compared to controls (209±30/120±38 vs. 151±12/88±17 mmHg, p<0.05) and serum catecholamine levels were elevated in HFpEF rats (38.4±5.6 vs. 25.6±1.5 ng/ml, p<0.05). Heart rate at peak exercise was decreased in HFpEF rats compared to controls (496±21 vs. 538±20 bpm, p<0.05) and exercise capacity was reduced in HFpEF rats (170±99 vs. 289±120 meters, p<0.05). Corrected SNRT was prolonged in HFpEF rats (86±34 vs. 58±10 ms, p<0.05), indicative of sinus node dysfunction. Ex vivo sinus node preparations showed decreased heart rate response to isoproterenol (119±12 vs. 163±27 bpm, p<0.05). Beta-1 receptors gene expression ( Adrb1 ) were not decreased in HFpEF sinus node (93% in HFpEF vs. 100% in control, p=ns) nor were L-type Ca channels gene expression ( Cacna1c ) (95% in HFpEF vs. 100% in control, p=ns). However, pacemaker channels gene expression ( Hcn4 ) showed a trend toward reduction in HFpEF sinus nodes (60% in HFpEF vs. 100% in control, p=0.1). Conclusions: Sinus node dysfunction underlies chronotropic incompetence in rats with HFpEF. Further investigation is warranted to elucidate the mechanism and design potential therapeutic strategies for this condition.

Disseminated tumour cells with highly aberrant genomes are linked to poor prognosis in operable oesophageal adenocarcinoma

Background:Chromosomal instability (CIN) has repeatedly been identified as a prognostic marker. Here we evaluated the percentage of aberrant genome per cell (PAG) as a measure of CIN in single disseminated tumour cells (DTC) isolated from patients with operable oesophageal adenocarcinoma (EAC), to assess the impact of CINhigh DTCs on prognosis.Methods:We isolated CK18positive DTCs from bone marrow (BM) or lymph node (LN) preparations of operable EAC patients. After whole-genome amplification, single DTCs were analysed for chromosomal gains and losses using metaphase-based comparative genomic hybridisation (mCGH). We calculated the PAG for each DTC and determined the critical threshold value that identifies high-risk patients by STEPP (Subpopulation Treatment Effect Pattern Plot) analysis in two independent EAC patient cohorts (cohort #1, n=44; cohort #2; n=29).Results:The most common chromosomal alterations observed among the DTCs were typical for EAC, but the DTCs showed a varying PAG between individual patients. Generally, LNDTCs displayed a significantly higher PAG than BMDTCs. STEPP analysis revealed an increasing PAG of DTCs to be correlated with an increased risk for short survival in two independent EAC cohorts as well as in the corresponding pooled analysis. In all three data sets (cohort #1, cohort #2 and pooled cohort), PAGhigh DTCs conferred an independent risk for a significantly decreased survival.Conclusions:The analysis of PAG/CIN in solitary marker-positive DTCs identifies operable EAC patients with poor prognosis, indicating a more aggressive minimal residual disease.

A retrospective analysis of lymph node status in advanced ovarian, fallopian, and peritoneal cancer.

e17074 Background: The purpose of this study was to assess lymph node (LN) status; greatest dimension (D), D of metastatic LN (LN (+)) foci (MF), number of LN (+) of each patient, and MF area aggregated in each patient affected by LN metastasis in FIGO stage III and IV ovarian, fallopian, and peritoneal cancer. Methods: We researched the consecutive 25 patients who underwent primary debulking surgery or neoadjuvant chemotherapy (NAC) followed by interval debulking surgery including systemic lymphadenectomy from 2004 to 2015. Non-serous histology was 20 % (5/25). LN (+) were detected in 12.6% (169/1344) of lymph node preparations. Results: The patients’ median age was 53 years. Median number of LN (+) and sum of MF area in each patient were 2 (range 0-37) and 2 mm2 (range 0- 498). Mean D of the entire LN and of the MF were 4.7mm (95%Confidence Interval (CI); 4.5,4.9) and 6.1mm(95% CI; 5.2, 7.0). The LN (+) displayed a significantly larger mean D value (7.9 mm, 95% CI: 7.4, 8.5) than the non-metastatic lymph nodes (LN (-)) (4.2 mm, 95% CI: 4.0, 4.4) (p<0.05; student t test). D of MF (mm) was approximated by LN (+) area (mm2) as follows; (D of MF) = 2.58+ 0.094*(LN (+) area)+ 0.062* ( ( LN (+) area)-43.2)2; R2=0.75; p<0.0001. The D of MF with NAC, 5.0 mm (95%CI: 3.6, 6.4) demonstrated smaller than that without NAC, 7.0 mm (95%CI: 5.8, 8.1) (p<0.05; student t test). The proportion of LN (-) patients was significantly higher in the NAC group (44%, 4/9) than in the non-NAC group (6%, 1/16) (p=0.022). D of LN (+), D of MF ≥10 mm, sum of MF area, and number of LN (+) ≥ 2 did not achieve statistical significance in analysis with the relative risk (RR) of death. Conclusions: LN (+) exhibits larger D than LN (-) and D of MF could be estimated by LN (+) area. Some MFs shrink after NAC, and hence, become difficult to detect pathologically. In advanced ovarian, fallopian, and peritoneal cancer LN size and sum of MF area could not show an impact on prognosis.

Ca(2+)-Clock-Dependent Pacemaking in the Sinus Node Is Impaired in Mice with a Cardiac Specific Reduction in SERCA2 Abundance.

Background: The sarcoplasmic reticulum Ca2+-ATPase (SERCA2) pump is an important component of the Ca2+-clock pacemaker mechanism that provides robustness and flexibility to sinus node pacemaking. We have developed transgenic mice with reduced cardiac SERCA2 abundance (Serca2 KO) as a model for investigating SERCA2's role in sinus node pacemaking.Methods and Results: In Serca2 KO mice, ventricular SERCA2a protein content measured by Western blotting was 75% (P < 0.05) lower than that in control mice (Serca2 FF) tissue. Immunofluorescent labeling of SERCA2a in ventricular, atrial, sinus node periphery and center tissue sections revealed 46, 45, 55, and 34% (all P < 0.05 vs. Serca2 FF) lower labeling, respectively and a mosaic pattern of expression. With telemetric ECG surveillance, we observed no difference in basal heart rate, but the PR-interval was prolonged in Serca2 KO mice: 49 ± 1 vs. 40 ± 1 ms (P < 0.001) in Serca2 FF. During exercise, heart rate in Serca2 KO mice was elevated to 667 ± 22 bpm, considerably less than 780 ± 17 bpm (P < 0.01) in Serca2 FF. In isolated sinus node preparations, 2 mM Cs+ caused bradycardia that was equally pronounced in Serca2 KO and Serca2 FF (32 ± 4% vs. 29 ± 5%), indicating no change in the pacemaker current, If. Disabling the Ca2+-clock with 2 μM ryanodine induced bradycardia that was less pronounced in Serca2 KO preparations (9 ± 1% vs. 20 ± 3% in Serca2 FF; P < 0.05), suggesting a disrupted Ca2+-clock. Mathematical modeling was used to dissect the effects of membrane- and Ca2+-clock components on Serca2 KO mouse heart rate and sinus node action potential. Computer modeling predicted a slowing of heart rate with SERCA2 downregulation and the heart rate slowing was pronounced at >70% reduction in SERCA2 activity.Conclusions: Serca2 KO mice show a disrupted Ca2+-clock-dependent pacemaker mechanism contributing to impaired sinus node and atrioventricular node function.

Contributions of cardiac "funny" (f) channels and sarcoplasmic reticulum Ca2+ in regulating beating rate of mouse and guinea pig sinoatrial node.

The aim of this study was to investigate the effects on spontaneous beating rate of mouse atrial preparations following selective block of cardiac "funny" (f) channels, I(f), and/or suppression of sarcoplasmic reticulum (SR) function in the absence and presence of β-adrenoceptor stimulation. ZD7288 [to block I(f)] caused a substantial reduction (222±13bpm) in beating rate from 431±14 to 209±14bpm, ryanodine alone (to block SR Ca(2+) release) reduced beating rate by 105±11bpm, with subsequent addition of ZD7288 further reducing rate by 57±9bpm. Cyclopiazonic acid (CPA) alone (to inhibit Ca(2+) reuptake by the SR) reduced beating rate by 148±13bpm with subsequent addition of ZD7288 further reducing rate by 79±12bpm. In additional experiments measuring Ca(2+) transients in the SA node region using Rhod-2, effects of ivabradine and ZD7288 on rate were again substantially reduced after CPA. Effects of CPA alone on rate developed much more slowly than effects on Ca(2+) transient amplitude. ZD7288, ivabradine, and CPA reduced the slope and maximum response of the log(concentration)-response curves for effects of isoprenaline on beating rate. Very little response to isoprenaline remained after treatment with CPA followed by ZD7288. Similar changes in isoprenaline log(concentration)-response curves were seen in guinea pig preparations. These observations are consistent with a role for Ca(2+) released from the SR in regulating I(f) and therefore beating rate of SA node preparations; there appear to be additional contributions of SR-derived Ca(2+) to effects of β-adrenoceptor stimulation on beating rate that are independent of I(f).

Selective and non-selective non-steroidal anti-inflammatory drugs differentially regulate pulmonary vein and atrial arrhythmogenesis

BackgroundNon-steroidal anti-inflammatory drugs (NSAIDs) increase the risk of atrial fibrillation (AF). This study investigated whether selective and non-selective NSAIDs differentially regulate the arrhythmogenesis of pulmonary veins and atria. MethodsConventional microelectrodes were used to record action potentials (APs) in isolated rabbit PVs, sinoatrial node (SAN), left atrium (LA), and right atrium (RA) preparations before and after celecoxib or indomethacin administration. A whole-cell patch clamp was used to record the sodium–calcium exchanger (NCX) current, L-type calcium current (ICa-L), and late sodium current (INa-late) before and after celecoxib administration in isolated PV cardiomyocytes. ResultsCelecoxib (0.3, 1, and 3μM) reduced PV spontaneous beating rates, and induced delayed afterdepolarizations and burst firings in four of eight PV preparations (50%, p<0.05). Celecoxib also reduced SAN beating rates and decreased AP durations (APDs) in RA and LA, but did not change the resting membrane potential. Indomethacin (0.3, 1, 3, and 10μM) changed neither the PV or SAN beating rates nor RA APDs, but it reduced LA APDs. Celecoxib (3μM) significantly increased the NCX current and decreased the ICa-L, but did not change the INa-late. Ranolazine (10μM) suppressed celecoxib (3μM)-induced PV burst firings in 6 (86%, p<0.05) of 7 PVs. KB-R7943 (10μM) suppressed celecoxib (3μM)-induced PV burst firings in 5 (71%, p<0.05) of 7 PVs. ConclusionsSelective and non-selective NSAIDs differentially modulate PV and atrial electrophysiological characteristics. Celecoxib increased PV triggered activity through enhancement of the NCX current, which contributed to its arrhythmogenesis.

Abstract 16515: Differential Functions of HCN1 and HCN4 Pacemaker Channels Within the Sinoatrial Node for the Stable and Precise Beating of the Heart

The heart beat is initiated by the generation of spontaneous action potentials in pacemaker cells of the sinoatrial node (SAN) region. The maintenance of a stable heart beat requires mechanisms which protect the SAN pacemaker cells from potential perturbing influences which arise from inside and outside the sinoatrial network. Our previous work suggests that the hyperpolarization-activated cyclic nucleotide gated channel subtype 1 (HCN1) protects against such perturbations and thereby increases the stability, the precision and the safety of the sinoatrial network. Here, we investigate the role of HCN4 channels within this context. Using genetic mouse models deficient for HCN channels as well as mouse models expressing engineered HCN channels, we performed a detailed functional characterization of pacemaker mechanisms in single isolated sinoatrial node cells, explanted beating sinoatrial node preparations, with telemetric in vivo electrocardiography, echocardiography, and in vivo electrophysiology. We provide evidence that HCN4 has a critical role in counteracting and balancing potentially destabilizing effects of the autonomic nervous system on the regulation of the heart rate. Specifically, HCN4 channels smooth the transition of the heart rate to a new equilibrium. Furthermore, we provide evidence that the cAMP- dependent fine tuning of HCN4 channel activity could provide the exact dosage of current to balance and counteract overshooting responses of the heart rate to autonomic regulation. In the absence of such a protecting effect, mice display a brady- tachy syndrome.

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

Sick Sinus Syndrome in HCN1-Deficient Mice

Sinus node dysfunction (SND) is a major clinically relevant disease that is associated with sudden cardiac death and requires surgical implantation of electric pacemaker devices. Frequently, SND occurs in heart failure and hypertension, conditions that lead to electric instability of the heart. Although the pathologies of acquired SND have been studied extensively, little is known about the molecular and cellular mechanisms that cause congenital SND. Here, we show that the HCN1 protein is highly expressed in the sinoatrial node and is colocalized with HCN4, the main sinoatrial pacemaker channel isoform. To characterize the cardiac phenotype of HCN1-deficient mice, a detailed functional characterization of pacemaker mechanisms in single isolated sinoatrial node cells, explanted beating sinoatrial node preparation, telemetric in vivo electrocardiography, echocardiography, and in vivo electrophysiology was performed. On the basis of these experiments we demonstrate that mice lacking the pacemaker channel HCN1 display congenital SND characterized by bradycardia, sinus dysrhythmia, prolonged sinoatrial node recovery time, increased sinoatrial conduction time, and recurrent sinus pauses. As a consequence of SND, HCN1-deficient mice display a severely reduced cardiac output. We propose that HCN1 stabilizes the leading pacemaker region within the sinoatrial node and hence is crucial for stable heart rate and regular beat-to-beat variation. Furthermore, we suggest that HCN1-deficient mice may be a valuable genetic disease model for human SND.