Effect of contact force on pulsed field ablation lesions in porcine cardiac tissue.

INTRODUCTION: Contact force has been used to titrate lesion formation for radiofrequency ablation. Pulsed field ablation (PFA) is a field-based ablation technology for which limited evidence on the impact of contact force on lesion size is available. METHODS: Porcine hearts (n = 6) were perfused using a modified Langendorff set-up. A prototype focal PFA catheter attached to a force gauge was held perpendicular to the epicardium and lowered until contact was made. Contact force was recorded during each PFA delivery. Matured lesions were cross-sectioned, stained, and the lesion dimensions measured. RESULTS: A total of 82 lesions were evaluated with contact forces between 1.3 and 48.6 g. Mean lesion depth was 4.8 ± 0.9 mm (standard deviation), mean lesion width was 9.1 ± 1.3 mm, and mean lesion volume was 217.0 ± 96.6 mm3 . Linear regression curves showed an increase of only 0.01 mm in depth (depth = 0.01 × contact force + 4.41, R2 = 0.05), 0.03 mm in width (width = 0.03 × contact force + 8.26, R2 = 0.13) for each additional gram of contact force, and 2.20 mm3 in volume (volume = 2.20 × contact force + 162, R2 = 0.10). CONCLUSION: Increasing contact force using a bipolar, biphasic focal PFA system has minimal effects on acute lesion dimensions in an isolated porcine heart model and achieving tissue contact is more important than the force with which that contact is made.

Microcanonical coarse-graining of the kinetic Ising model.

We propose a scheme for coarse-graining the dynamics of the 2-D kinetic Ising model onto the microcanonical ensemble. At subcritical temperatures, 2-D and higher-dimensional Ising lattices possess two basins of attraction separated by a free energy barrier. Projecting onto the microcanonical ensemble has the advantage that the dependence of the crossing rate constant on environmental conditions can be obtained from a single Monte Carlo trajectory. Using various numerical methods, we computed the forward rate constants of coarse-grained representations of the Ising model and compared them with the true value obtained from brute force simulation. While coarse-graining preserves detailed balance, the computed rate constants for barrier heights between 5 kT and 9 kT were consistently 50% larger than the true value. Markovianity testing revealed loss of dynamical memory, which we propose accounts for coarse-graining error. Committor analysis did not support the alternative hypothesis that microcanonical projection is incompatible with an optimal reaction coordinate. The correct crossing rate constant was obtained by spectrally decomposing the diffusion coefficient near the free energy barrier and selecting the slowest (reactive) component. The spectral method also yielded the correct rate constant in the 3-D Ising lattice, where coarse-graining error was 6% and memory effects were diminished. We conclude that microcanonical coarse-graining supplemented by spectral analysis of short-term barrier fluctuations provides a comprehensive kinetic description of barrier crossing in a non-inertial continuous-time jump process.

Prospects for Detecting Gravitational Waves at 5 Hz with Ground-Based Detectors.

We propose an upgrade to Advanced LIGO (aLIGO), named LIGO-LF, that focuses on improving the sensitivity in the 5-30 Hz low-frequency band, and we explore the upgrade's astrophysical applications. We present a comprehensive study of the detector's technical noises and show that with technologies currently under development, such as interferometrically sensed seismometers and balanced-homodyne readout, LIGO-LF can reach the fundamental limits set by quantum and thermal noises down to 5 Hz. These technologies are also directly applicable to the future generation of detectors. We go on to consider this upgrade's implications for the astrophysical output of an aLIGO-like detector. A single LIGO-LF can detect mergers of stellar-mass black holes (BHs) out to a redshift of z≃6 and would be sensitive to intermediate-mass black holes up to 2000 M_{⊙}. The detection rate of merging BHs will increase by a factor of 18 compared to aLIGO. Additionally, for a given source the chirp mass and total mass can be constrained 2 times better than aLIGO and the effective spin 3-5 times better than aLIGO. Furthermore, LIGO-LF enables the localization of coalescing binary neutron stars with an uncertainty solid angle 10 times smaller than that of aLIGO at 30 Hz and 4 times smaller when the entire signal is used. LIGO-LF also significantly enhances the probability of detecting other astrophysical phenomena including the tidal excitation of neutron star r modes and the gravitational memory effects.

Functional heterogeneity of the four voltage sensors of a human L-type calcium channel.

Excitation-evoked Ca(2+) influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca(2+) entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I-IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.

Observation of Parametric Instability in Advanced LIGO.

Parametric instabilities have long been studied as a potentially limiting effect in high-power interferometric gravitational wave detectors. Until now, however, these instabilities have never been observed in a kilometer-scale interferometer. In this Letter, we describe the first observation of parametric instability in a gravitational wave detector, and the means by which it has been removed as a barrier to progress.

Cardiac I-1c overexpression with reengineered AAV improves cardiac function in swine ischemic heart failure.

Cardiac gene therapy has emerged as a promising option to treat advanced heart failure (HF). Advances in molecular biology and gene targeting approaches are offering further novel options for genetic manipulation of the cardiovascular system. The aim of this study was to improve cardiac function in chronic HF by overexpressing constitutively active inhibitor-1 (I-1c) using a novel cardiotropic vector generated by capsid reengineering of adeno-associated virus (BNP116). One month after a large anterior myocardial infarction, 20 Yorkshire pigs randomly received intracoronary injection of either high-dose BNP116.I-1c (1.0 × 10(13) vector genomes (vg), n = 7), low-dose BNP116.I-1c (3.0 × 10(12) vg, n = 7), or saline (n = 6). Compared to baseline, mean left ventricular ejection fraction increased by 5.7% in the high-dose group, and by 5.2% in the low-dose group, whereas it decreased by 7% in the saline group. Additionally, preload-recruitable stroke work obtained from pressure-volume analysis demonstrated significantly higher cardiac performance in the high-dose group. Likewise, other hemodynamic parameters, including stroke volume and contractility index indicated improved cardiac function after the I-1c gene transfer. Furthermore, BNP116 showed a favorable gene expression pattern for targeting the heart. In summary, I-1c overexpression using BNP116 improves cardiac function in a clinically relevant model of ischemic HF.

Self-amplified lock of an ultra-narrow linewidth optical cavity.

High finesse optical cavities are an essential tool in modern precision laser interferometry. The incident laser field is often controlled and stabilized with an active feedback system such that the field resonates in the cavity. The Pound-Drever-Hall reflection locking technique is a convenient way to derive a suitable error signal. However, it only gives a strong signal within the cavity linewidth. This poses a problem for locking an ultra-narrow linewidth cavity. We present a novel technique for acquiring lock by utilizing an additional weak control signal, but with a much larger capture range. We numerically show that this technique can be applied to the laser frequency stabilization system used in the Laser Interferometric Gravitational-wave Observatory (LIGO), which has a linewidth of 0.8 Hz. This new technique will allow us to robustly and repeatedly lock the LIGO laser frequency to the common mode of the interferometer.

The importance of electrode-tissue proximity in creating pulsed field ablation lesions: insights from a sub-acute preclinical model.

BACKGROUND: We sought to evaluate the anatomic and functional lesion development over time at different atrial sites immediately following delivery of pulsed field ablation (PFA). METHODS: Using a porcine model, PFA ablations were performed in the superior vena cava (SVC), right atrial lateral wall (RA), left atrial appendage (LAA), and right superior pulmonary vein (RSPV) using four different PFA profiles. Mapping was done sequentially in 5-20-min increments up to 280-min post lesion delivery for low voltage area (LVA) assessment and conduction velocity. Lesion characteristics were noted with voltage mapping immediately post ablation and at the serial time points. RESULTS: In 9 animals, 33 sites were ablated. None of the four different profiles across all sites showed any statistical difference on acute lesion formation or persistence. Higher tissue contact was observed in the SVC and RSPV and lower tissue contact was observed in the LAA and RA locations. Higher contact areas were noted to have higher density electroanatomic low voltage area (LVA) (12/14 vs 5/18, p = 0.01) and larger lesions on gross pathology (2 /14 vs 6/16, p = 0.01) compared to lower contact areas. Lesion regression occurred in 16/33 sites. Sustained lesions were significantly more prevalent in higher versus lower contact sites (65% vs 38%, p = 0.037). CONCLUSION: The development of significant and durable lesions for PFA in a porcine model appears to be dependent on tissue proximity and contact.

The contribution of RCK domains to human BK channel allosteric activation.

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca(2+)-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca(2+)-sensing apparatus known as the "gating ring." The allosteric interplay between voltage- and Ca(2+)-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca(2+), we optically resolved VSD activation prompted by Ca(2+) binding to the gating ring. The sudden increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca(2+) sensor located in the RCK2 domain abolished the effect of [Ca(2+)](i) increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca(2+) sensing did not prevent the effect of Ca(2+) release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca(2+) association in two distinct sites, voltage sensor activation, and BK channel opening.

Operation of the voltage sensor of a human voltage- and Ca2+-activated K+ channel.

Voltage sensor domains (VSDs) are structurally and functionally conserved protein modules that consist of four transmembrane segments (S1-S4) and confer voltage sensitivity to many ion channels. Depolarization is sensed by VSD-charged residues residing in the membrane field, inducing VSD activation that facilitates channel gating. S4 is typically thought to be the principal functional component of the VSD because it carries, in most channels, a large portion of the VSD gating charge. The VSDs of large-conductance, voltage- and Ca(2+)-activated K(+) channels are peculiar in that more gating charge is carried by transmembrane segments other than S4. Considering its "decentralized" distribution of voltage-sensing residues, we probed the BK(Ca) VSD for evidence of cooperativity between charge-carrying segments S2 and S4. We achieved this by optically tracking their activation by using voltage clamp fluorometry, in channels with intact voltage sensors and charge-neutralized mutants. The results from these experiments indicate that S2 and S4 possess distinct voltage dependence, but functionally interact, such that the effective valence of one segment is affected by charge neutralization in the other. Statistical-mechanical modeling of the experimental findings using allosteric interactions demonstrates two mechanisms (mechanical coupling and dynamic focusing of the membrane electric field) that are compatible with the observed cross-segment effects of charge neutralization.

Determination of lethal electric field threshold for pulsed field ablation in ex vivo perfused porcine and human hearts.

Introduction: Pulsed field ablation is an emerging modality for catheter-based cardiac ablation. The main mechanism of action is irreversible electroporation (IRE), a threshold-based phenomenon in which cells die after exposure to intense pulsed electric fields. Lethal electric field threshold for IRE is a tissue property that determines treatment feasibility and enables the development of new devices and therapeutic applications, but it is greatly dependent on the number of pulses and their duration. Methods: In the study, lesions were generated by applying IRE in porcine and human left ventricles using a pair of parallel needle electrodes at different voltages (500-1500 V) and two different pulse waveforms: a proprietary biphasic waveform (Medtronic) and monophasic 48 × 100 µs pulses. The lethal electric field threshold, anisotropy ratio, and conductivity increase by electroporation were determined by numerical modeling, comparing the model outputs with segmented lesion images. Results: The median threshold was 535 V/cm in porcine ((N = 51 lesions in n = 6 hearts) and 416 V/cm in the human donor hearts ((N = 21 lesions in n = 3 hearts) for the biphasic waveform. The median threshold value was 368 V/cm in porcine hearts ((N = 35 lesions in n = 9 hearts) cm for 48 × 100 µs pulses. Discussion: The values obtained are compared with an extensive literature review of published lethal electric field thresholds in other tissues and were found to be lower than most other tissues, except for skeletal muscle. These findings, albeit preliminary, from a limited number of hearts suggest that treatments in humans with parameters optimized in pigs should result in equal or greater lesions.

Characterization of Experimentally Observed Complex Interplay between Pulse Duration, Electrical Field Strength, and Cell Orientation on Electroporation Outcome Using a Time-Dependent Nonlinear Numerical Model.

Electroporation is a biophysical phenomenon involving an increase in cell membrane permeability to molecules after a high-pulsed electric field is applied to the tissue. Currently, electroporation is being developed for non-thermal ablation of cardiac tissue to treat arrhythmias. Cardiomyocytes have been shown to be more affected by electroporation when oriented with their long axis parallel to the applied electric field. However, recent studies demonstrate that the preferentially affected orientation depends on the pulse parameters. To gain better insight into the influence of cell orientation on electroporation with different pulse parameters, we developed a time-dependent nonlinear numerical model where we calculated the induced transmembrane voltage and pores creation in the membrane due to electroporation. The numerical results show that the onset of electroporation is observed at lower electric field strengths for cells oriented parallel to the electric field for pulse durations ≥10 µs, and cells oriented perpendicular for pulse durations ~100 ns. For pulses of ~1 µs duration, electroporation is not very sensitive to cell orientation. Interestingly, as the electric field strength increases beyond the onset of electroporation, perpendicular cells become more affected irrespective of pulse duration. The results obtained using the developed time-dependent nonlinear model are corroborated by in vitro experimental measurements. Our study will contribute to the process of further development and optimization of pulsed-field ablation and gene therapy in cardiac treatments.

A Multiscale Computational Model of Skeletal Muscle Electroporation Validated Using In Situ Porcine Experiments.

OBJECTIVE: The goal of our study was to determine the importance of electric field orientation in an anisotropic muscle tissue for the extent of irreversible electroporation damage by means of an experimentally validated mathematical model. METHODS: Electrical pulses were delivered to porcine skeletal muscle in vivo by inserting needle electrodes so that the electric field was applied in direction either parallel or perpendicular to the direction of the muscle fibres. Triphenyl tetrazolium chloride staining was used to determine the shape of the lesions. Next, we used a single cell model to determine the cell-level conductivity during electroporation, and then generalised the calculated conductivity changes to the bulk tissue. Finally, we compared the experimental lesions with the calculated field strength distributions using the Sørensen-Dice similarity coefficient to find the contours of the electric field strength threshold beyond which irreversible damage is thought to occur. RESULTS: Lesions in the parallel group were consistently smaller and narrower than lesions in the perpendicular group. The determined irreversible threshold of electroporation for the selected pulse protocol was 193.4 V/cm with a standard deviation of 42.1 V/cm, and was not dependent on field orientation. CONCLUSION: Muscle anisotropy is of significant importance when considering electric field distribution in electroporation applications. SIGNIFICANCE: The paper presents an important advancement in building up from the current understanding of single cell electroporation to an in silico multiscale model of bulk muscle tissue. The model accounts for anisotropic electrical conductivity and has been validated through experiments in vivo.

The Effects of Interphase and Interpulse Delays and Pulse Widths on Induced Muscle Contractions, Pain and Therapeutic Efficacy in Electroporation-Based Therapies.

Electroporation is used in medicine for drug and gene delivery, and as a nonthermal ablation method in tumor treatment and cardiac ablation. Electroporation involves delivering high-voltage electric pulses to target tissue; however, this can cause effects beyond the intended target tissue like nerve stimulation, muscle contractions and pain, requiring use of sedatives or anesthetics. It was previously shown that adjusting pulse parameters may mitigate some of these effects, but not how these adjustments would affect electroporation's efficacy. We investigated the effect of varying pulse parameters such as interphase and interpulse delay while keeping the duration and number of pulses constant on nerve stimulation, muscle contraction and assessing pain and electroporation efficacy, conducting experiments on human volunteers, tissue samples and cell lines in vitro. Our results show that using specific pulse parameters, particularly short high-frequency biphasic pulses with short interphase and long interpulse delays, reduces muscle contractions and pain sensations in healthy individuals. Higher stimulation thresholds were also observed in experiments on isolated swine phrenic nerves and human esophagus tissues. However, changes in the interphase and interpulse delays did not affect the cell permeability and survival, suggesting that modifying the pulse parameters could minimize adverse effects while preserving therapeutic goals in electroporation.

Arm-length stabilisation for interferometric gravitational-wave detectors using frequency-doubled auxiliary lasers.

Residual motion of the arm cavity mirrors is expected to prove one of the principal impediments to systematic lock acquisition in advanced gravitational-wave interferometers. We present a technique which overcomes this problem by employing auxiliary lasers at twice the fundamental measurement frequency to pre-stabilise the arm cavities' lengths. Applying this approach, we reduce the apparent length noise of a 1.3 m long, independently suspended Fabry-Perot cavity to 30 pm rms and successfully transfer longitudinal control of the system from the auxiliary laser to the measurement laser.

Reversible and Irreversible Effects of Electroporation on Contractility and Calcium Homeostasis in Isolated Cardiac Ventricular Myocytes.

BACKGROUND: Irreversible electroporation is an energy form utilizing high-voltage pulsed electric field, leading to cellular homeostasis disruption and cell death. Recently, irreversible electroporation has shown promising results for the treatment of cardiac arrhythmias. However, reversible and irreversible effects of pulsed electric field on cardiac myocytes remain poorly understood. Here, we evaluated the influence of a monophasic single electric pulse (EP) on the contractility, Ca2+ homeostasis and recovery of cardiac myocytes. METHODS: Isolated rat left ventricular myocytes were electroporated using single monophasic EP of different durations and voltages. Sarcomere length and intracellular Ca2+ were simultaneously monitored for up to 20 minutes after EP application in Fura-2 loaded left ventricular myocytes. Lethal voltage thresholds were determined using 100 µs and 10 ms pulses and by discriminating cell orientation with respect to the electric field. RESULTS: Electroporation led to an immediate increase in intracellular Ca2+ which was dependent upon the voltage delivered to the cell. Intermediate-voltage EP (140 V, 100 µs) increased sarcomere shortening, Ca2+ transient amplitude, and diastolic Ca2+ level measured 1 minute post-EP. Although sarcomere shortening returned to pre-EP level within 5 minutes, Ca2+ transient amplitude decreased further below pre-EP level and diastolic Ca2+ level remained elevated within 20 minutes post-EP. Spontaneous contractions were observed after sublethal EP application but their frequency decreased progressively within 20 minutes. Lethal EP voltage threshold was lower in myocytes oriented perpendicular than parallel to the electric field using 100 µs pulses while an opposite effect was found using 10 ms pulses. CONCLUSIONS: Sublethal EP affected rat left ventricular myocytes contractility and disrupted Ca2+ homeostasis as a function of the EP voltage. Moreover, EP-induced lethality was preceded by a large increase in intracellular Ca2+ and was dependent upon the EP duration, amplitude and left ventricular myocytes orientation with respect to the electric field. These findings provide new insights into the effect of pulsed electric field on cardiac myocytes.

Characterization of Phrenic Nerve Response to Pulsed Field Ablation.

BACKGROUND: Phrenic nerve palsy is a well-known complication of cardiac ablation, resulting from the application of direct thermal energy. Emerging pulsed field ablation (PFA) may reduce the risk of phrenic nerve injury but has not been well characterized. METHODS: Accelerometers and continuous pacing were used during PFA deliveries in a porcine model. Acute dose response was established in a first experimental phase with ascending PFA intensity delivered to the phrenic nerve (n=12). In a second phase, nerves were targeted with a single ablation level to observe the effect of repetitive ablations on nerve function (n=4). A third chronic phase characterized assessed histopathology of nerves adjacent to ablated cardiac tissue (n=6). RESULTS: Acutely, we observed a dose-dependent response in phrenic nerve function including reversible stunning (R2=0.965, P<0.001). Furthermore, acute results demonstrated that phrenic nerve function responded to varying levels of PFA and catheter proximity placements, resulting in either: no effect, effect, or stunning. In the chronic study phase, successful isolation of superior vena cava at a dose not predicted to cause phrenic nerve dysfunction was associated with normal phrenic nerve function and normal phrenic nerve histopathology at 4 weeks. CONCLUSIONS: Proximity of the catheter to the phrenic nerve and the PFA dose level were critical for phrenic nerve response. Gross and histopathologic evaluation of phrenic nerves and diaphragms at a chronic time point yielded no injury. These results provide a basis for understanding the susceptibility and recovery of phrenic nerves in response to PFA and a need for appropriate caution in moving beyond animal models.

Effects of Electrode-Tissue Proximity on Cardiac Lesion Formation Using Pulsed Field Ablation.

BACKGROUND: Pulsed field ablation (PFA) is a novel energy modality for treatment of cardiac arrhythmias. The impact of electrode-tissue proximity on lesion formation by PFA has not been conclusively assessed. The objective of this investigation was to evaluate the effects of electrode-tissue proximity on cardiac lesion formation with a biphasic, bipolar PFA system. METHODS: PFA was delivered on the ventricular epicardial surface in an isolated porcine heart model (n=8) via a 4-electrode prototype catheter. An offset tool was designed to control the distance between electrodes and target tissue; deliveries were placed 0 mm (0 mm offset), 2 mm (2 mm offset), and 4 mm away from the tissue (4 mm offset). Lesions were assessed using tetrazolium chloride staining. Numerical models for the experimental setup with and without the offset tool validated and supported results. RESULTS: Cardiac lesion dimensions decreased proportional to the distance between epicardial surface and electrodes. Lesion depth averaged 4.3±0.4 mm, 2.7±0.4 mm, and 1.3±0.4 mm for the 0, 2, and 4 mm and lesion width averaged 9.4±1.1 mm, 7.5±0.8 mm and 5.8±1.4 mm for the 0, 2, and 4 mm offset distances, respectively. Numerical modeling matched ex vivo results well and predicted lesion creation with and without the offset tool. CONCLUSIONS: Using a biphasic, bipolar PFA system resulted in cardiac lesions even in the 0 mm offset distance case. The relationship between lesion depth and offset distance was linear, and the deepest lesions were created with 0 mm offset distance, that is, with electrodes in contact with tissue. Therefore, close electrode-tissue proximity increases the likelihood of achieving transmural lesions by maximizing the electric field penetration into the target tissue.

Glucagon-like peptide-1 enhances cardiac L-type Ca2+ currents via activation of the cAMP-dependent protein kinase A pathway.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is a hormone predominately synthesized and secreted by intestinal L-cells. GLP-1 modulates multiple cellular functions and its receptor agonists are now used clinically for diabetic treatment. Interestingly, preclinical and clinical evidence suggests that GLP-1 agonists produce beneficial effects on dysfunctional hearts via acting on myocardial GLP-1 receptors. As the effects of GLP-1 on myocyte electrophysiology are largely unknown, this study was to assess if GLP-1 could affect the cardiac voltage-gated L-type Ca2+ current (I(Ca)). METHODS: The whole-cell patch clamp method was used to record I(Ca) and action potentials in enzymatically isolated cardiomyocytes from adult canine left ventricles. RESULTS: Extracellular perfusion of GLP-1 (7-36 amide) at 5 nM increased I(Ca) by 23 ± 8% (p < 0.05, n = 7). Simultaneous bath perfusion of 5 nM GLP-1 plus 100 nM Exendin (9-39), a GLP-1 receptor antagonist, was unable to block the GLP-1-induced increase in I(Ca); however, the increase in I(Ca) was abolished if Exendin (9-39) was pre-applied 5 min prior to GLP-1 administration. Intracellular dialysis with a protein kinase A inhibitor also blocked the GLP-1-enhanced I(Ca). In addition, GLP-1 at 5 nM prolonged the durations of the action potentials by 128 ± 36 ms (p < 0.01) and 199 ± 76 ms (p < 0.05) at 50% and 90% repolarization (n = 6), respectively. CONCLUSIONS: Our data demonstrate that GLP-1 enhances I(Ca) in canine cardiomyocytes. The enhancement of I(Ca) is likely via the cAMP-dependent protein kinase A mechanism and may contribute, at least partially, to the prolongation of the action potential duration.

Development of an ovine model of pediatric complete heart block.

BACKGROUND: Complete heart block is a significant clinical problem that can limit the quality of life in affected children. To understand the pathophysiology of this condition and provide for development of novel therapies, we sought to establish a large animal model of permanent, pacemaker-dependent atrioventricular block (AVB) that mimics the size and growth characteristics of pediatric patients. MATERIALS AND METHODS: We utilized nine immature lambs weighing 10.5 ± 1.4 kg. After implantation of dual-chamber pacemaker devices with fixed leads, AVB was produced by interrupting His-bundle conduction using radio-frequency ablation at the base of the non-coronary cusp of the aortic valve. Ablations (30 to 60 s in duration) were performed under fluoroscopic guidance with electrophysiological monitoring. Interrogation of pacemakers and electrocardiography (ECG) determined the persistence of heart block. Ovine hearts were also examined immunohistochemically for localization of conduction tissue. RESULTS: AVB was produced in eight animals using an atypical approach from the left side of the heart. One animal died due to ventricular fibrillation during ablation proximal to the tricuspid annulus and one lamb was sacrificed postoperatively due to stroke. Four sheep were kept for long-term follow-up (109.8 ± 32.9 d) and required continuous ventricular pacing attributable to lasting AVB, despite significant increases in body weight and size. CONCLUSIONS: We have created a large animal model of pediatric complete heart block that is stable and technically practicable. We anticipate that this lamb model will allow for advancement of cell-based and other innovative treatments to repair complete heart block in children.