Characteristics of Radiofrequency Catheter Ablation Lesion Formation in Real Time In Vivo Using Near Field Ultrasound Imaging.

OBJECTIVES: Visualizing myocardium with near field ultrasound (NFUS) transducers in the tip of the catheter might provide an image of the evolving pathological lesion during energy delivery. BACKGROUND: Radiofrequency (RF) catheter ablation has been effective in arrhythmia treatment, but no technology has allowed lesion formation to be visualized in real time in vivo. METHODS: RF catheter ablations were performed in vivo with the goal to create transmural atrial lesions and large ventricular lesions. RF lesion formation was imaged in real time using M-mode, tissue Doppler, and strain rate information from the NFUS open irrigated RF ablation catheter incorporating 4 ultrasound transducers (1 axial and 3 radial), and growth kinetics were analyzed. Nineteen dogs underwent ablation in the right and left atria (n = 185), right ventricle (n = 67), and left ventricle (n = 66). Lesions were echolucent with tissue strain rate by NFUS. RESULTS: Lesion growth frequently progressed from epicardium to endocardium in thin-walled tissue. The half time of lesion growth was 5.5 ± 2.8 s in thin-walled and 9.7 ± 4.3 s in thick-walled tissue. Latency of lesion onset was seen in 57% of lesions ranging from 1 to 63.8 s. Tissue edema (median 25% increased wall thickness) formed immediately upon lesion formation in 83%, and intramyocardial steam was seen in 71% of cases. CONCLUSIONS: NFUS was effective in imaging RF catheter ablation lesion formation in real time. It was useful in assessing the dynamics of lesion growth and could visualize impending steam pops. It may be a useful technology to improve both safety and efficacy of RF catheter ablation.

Near-Field Ultrasound Imaging During Radiofrequency Catheter Ablation: Tissue Thickness and Epicardial Wall Visualization and Assessment of Radiofrequency Ablation Lesion Formation and Depth.

BACKGROUND: Safe and successful radiofrequency catheter ablation depends on creation of transmural lesions without collateral injury to contiguous structures. Near-field ultrasound (NFUS) imaging through transducers in the tip of an ablation catheter may provide important information about catheter contact, wall thickness, and ablation lesion formation. METHODS AND RESULTS: NFUS imaging was performed using a specially designed open-irrigated radiofrequency ablation catheter incorporating 4 ultrasound transducers. Tissue/phantom thickness was measured in vitro with varying contact angles. In vivo testing was performed in 19 dogs with NFUS catheters positioned in 4 chambers. Wall thickness measurements were made at 222 sites (excluding the left ventricle) and compared with measurements from intracardiac echocardiography. Imaging was used to identify the epicardium with saline infusion into the pericardial space at 39 sites. In vitro, the measured exceeded actual tissue/phantom thickness by 13% to 20%. In vivo, NFUS reliably visualized electrode-tissue contact, but sensitivity of epicardial imaging was 92%. The chamber wall thickness measured by NFUS correlated well with intracardiac echocardiography (r=0.86; P<0.0001). Sensitivity of lesion identification by NFUS was 94% for atrial and 95% for ventricular ablations. NFUS was the best parameter to predict lesion depth in right and left ventricle (r=0.47; P<0.0001; multiple regression P=0.0025). Lesion transmurality was correctly identified in 87% of atrial lesions. CONCLUSIONS: NFUS catheter imaging reliably assesses electrode-tissue contact and wall thickness. Its use during radiofrequency catheter ablation may allow the operator to assess the depth of ablation required for transmural lesion formation to optimize power delivery.

Tachycardia-induced silencing of subcellular Ca2+ signaling in atrial myocytes.

Atrial fibrillation (AF) is characterized by sustained high atrial activation rates and arrhythmogenic cellular Ca2+ signaling instability; however, it is not clear how a high atrial rate and Ca2+ instability may be related. Here, we characterized subcellular Ca2+ signaling after 5 days of high atrial rates in a rabbit model. While some changes were similar to those in persistent AF, we identified a distinct pattern of stabilized subcellular Ca2+ signaling. Ca2+ sparks, arrhythmogenic Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and SR Ca2+ content were largely unaltered. Based on computational analysis, these findings were consistent with a higher Ca2+ leak due to PKA-dependent phosphorylation of SR Ca2+ channels (RyR2s), fewer RyR2s, and smaller RyR2 clusters in the SR. We determined that less Ca2+ release per [Ca2+]i transient, increased Ca2+ buffering strength, shortened action potentials, and reduced L-type Ca2+ current contribute to a stunning reduction of intracellular Na+ concentration following rapid atrial pacing. In both patients with AF and in our rabbit model, this silencing led to failed propagation of the [Ca2+]i signal to the myocyte center. We conclude that sustained high atrial rates alone silence Ca2+ signaling and do not produce Ca2+ signaling instability, consistent with an adaptive molecular and cellular response to atrial tachycardia.

Visualizing intramyocardial steam formation with a radiofrequency ablation catheter incorporating near-field ultrasound.

INTRODUCTION: Steam pops are a risk of irrigated RF ablation even when limiting power delivery. There is currently no way to predict gas formation during ablation. It would be useful to visualize intramyocardial gas formation prior to a steam pop occurring using near-field ultrasound integrated into a RF ablation catheter. METHODS AND RESULTS: In an in vivo open-chest ovine model (n = 9), 86 lesions were delivered to the epicardial surface of the ventricles. Energy was delivered for 15-60 seconds, to achieve lesions with and without steam pops, based on modeling data. The ultrasound image was compared to a digital audio recording from within the pericardium by a blinded observer. Of 86 lesions, 28 resulted in an audible steam pop. For lesions that resulted in a steam pop compared to those that did not (n = 58), the mean power delivered was 8.0 ± 1.8 W versus 6.7 ± 2.0 W, P = 0.006. A change in US contrast due to gas formation in the tissue occurred in all lesions that resulted in a steam pop. In 4 ablations, a similar change in US contrast was observed in the tissue and RF delivery was stopped; in these cases, no pop occurred. The mean depth of gas formation was 0.9 ± 0.8 mm, which correlated with maximal temperature predicted by modeling. Changes in US contrast occurred 7.6 ± 7.2 seconds before the impedance rise and 7.9 ± 6.2 seconds (0.1-17.0) before an audible pop. CONCLUSION: Integrated US in an RF ablation catheter is able to visualize gas formation intramyocardially several seconds prior to a steam pop occurring. This technology may help prevent complications arising from steam pops.

Real-time lesion assessment using a novel combined ultrasound and radiofrequency ablation catheter.

BACKGROUND: Assessment of lesion size and transmurality is currently via indirect measures. Real-time image assessment may allow ablation parameters to be titrated to achieve transmurality and reduce recurrences due to incomplete lesions. OBJECTIVE: The purpose of this study was to visualize lesion formation in real time using a novel combined ultrasound and externally irrigated ablation catheter. METHODS: In an in vivo open-chest sheep model, 144 lesions were delivered in 11 sheep to both the atria and the ventricles, while lesion development was monitored in real time. Energy was delivered for a minimum of 15 seconds and a maximum of 60 seconds, with a range of powers, to achieve different lesion depths. Twenty-two lesions were also delivered endocardially. The ultrasound appearance was assessed and compared with the pathological appearance by four independent blinded observers. RESULTS: For the ventricular lesions (n = 126), the mean power delivered was 6.1 ± 2.0 W, with a mean impedance of 394.7 ± 152.4 Ω and with an impedance drop of 136.4 ± 100.1 Ω. Lesion depths varied from 0 to 10 mm, with a median depth of 3.5 mm. At tissue depths up to 5 mm, changes in ultrasound contrast correlated well (r = 0.79, R(2) = 0.62) with tissue necrosis. The depth of ultrasound contrast correlated poorly with the depth of the zone of hemorrhage (r = 0.33, R(2) = 0.11), and impedance change correlated poorly with lesion depth (r = 0.29, R(2) = 0.08). CONCLUSION: Real-time lesion assessment using high-frequency ultrasound integrated into an ablation catheter is feasible and allows differentiation between true necrosis and hemorrhage. This may lead to safer and more efficient power delivery, allowing more effective lesion formation.

Distinct contractile and molecular differences between two goat models of atrial dysfunction: AV block-induced atrial dilatation and atrial fibrillation.

Atrial dilatation is an independent risk factor for thromboembolism in patients with and without atrial fibrillation (AF). In many patients, atrial dilatation goes along with depressed contractile function of the dilated atria. While some mechanisms causing atrial contractile dysfunction in fibrillating atria have been addressed previously, the cellular and molecular mechanisms of atrial contractile remodeling in dilated atria are unknown. This study characterized in vivo atrial contractile function in a goat model of atrial dilatation and compared it to a goat model of AF. Differences in the underlying mechanisms were elucidated by studying contractile function, electrophysiology and sarcoplasmic reticulum (SR) Ca2+ load in atrial muscle bundles and by analyzing expression and phosphorylation levels of key Ca2+-handling proteins, myofilaments and the expression and activity of their upstream regulators. In 7 chronically instrumented, awake goats atrial contractile dysfunction was monitored during 3 weeks of progressive atrial dilatation after AV-node ablation (AV block goats (AVB)). In open chest experiments atrial work index (AWI) and refractoriness were measured (10 goats with AVB, 5 goats with ten days of AF induced by repetitive atrial burst pacing (AF), 10 controls). Isometric force of contraction (FC), transmembrane action potentials (APs) and rapid cooling contractures (RCC, a measure of SR Ca2+ load) were studied in right atrial muscle bundles. Total and phosphorylated Ca2+-handling and myofilament protein levels were quantified by Western blot. In AVB goats, atrial size increased by 18% (from 26.6+/-4.4 to 31.6+/-5.5 mm, n=7 p<0.01) while atrial fractional shortening (AFS) decreased (from 18.4+/-1.7 to 12.8+/-4.0% at 400 ms, n=7, p<0.01). In open chest experiments, AWI was reduced in AVB and in AF goats compared to controls (at 400 ms: 8.4+/-0.9, n=7, and 3.2+/-1.8, n=5, vs 18.9+/-5.3 mmxmmHg, n=7, respectively, p<0.05 vs control). FC of isolated right atrial muscle bundles was reduced in AVB (n=8) and in AF (n=5) goats compared to controls (n=9) (at 2 Hz: 2.3+/-0.5 and 0.7+/-0.2 vs 5.5+/-1.0 mN/mm2, respectively, p<0.05). APs were shorter in AF, but unchanged in AVB goats. RCCs were reduced in AVB and AF versus control (AVB, 3.4+/-0.5 and AF, 4.1+/-1.4 vs 12.2+/-3.2 mN/mm2, p<0.05). Protein levels of protein kinase A (PKA) phosphorylated phospholamban (PLB) were reduced in AVB (n=8) and AF (n=8) vs control (n=7) by 37.9+/-12.4% and 29.7+/-10.1%, respectively (p<0.01), whereas calmodulin-dependent protein kinase II (CaMKII) phosphorylated ryanodine channels (RyR2) were increased by 166+/-55% in AVB (n=8) and by 146+/-56% in AF (n=8) goats (p<0.01). PKA-phosphorylated myosin-binding protein-C and troponin-I were reduced exclusively in AVB goat atria (by 75+/-10% and 55+/-15%, respectively, n=8, p<0.05). Atrial dilatation developing during slow ventricular rhythm after complete AV block as well as AF-induced remodeling are associated with atrial contractile dysfunction. Both AVB and AF goat atria show decreased SR Ca2+ load, likely caused by PLB dephosphorylation and RYR2 hyperphosphorylation. While shorter APs further compromise contractility in AF goat atria, reduced myofilament phosphorylation may impair contractility in AVB goat atria. Thus, atrial hypocontractility appears to have distinct molecular contributors in different types of atrial remodeling.

Blockade of atrial-specific K+-currents increases atrial but not ventricular contractility by enhancing reverse mode Na+/Ca2+-exchange.

BACKGROUND: AVE0118 (2'-{[2-(4-Methoxy-phenyl)-acetylamino]-methyl}-biphenyl-2-carboxylic acid (2-pyridin-3-yl-ethyl)-amide) blocks atrial ultrarapid delayed rectifier currents (I(Kur)) and prolongs the atrial action potential (AP) plateau without affecting ventricular repolarisation. In patients with atrial contractile dysfunction due to atrial tachyarrhythmias, this response might increase atrial contractility without risk of ventricular proarrhythmia. This study was designed to evaluate the inotropic mechanisms of AVE0118. METHODS AND RESULTS: In isometrically contracting atrial trabeculae, AVE0118 increased contractile force by 55.4% in sinus rhythm patients (n = 9) and by 107.4% in patients with atrial fibrillation (n = 8). In freshly isolated canine atrial myocytes studied under perforated patch current clamp (37 degrees C), AVE0118 increased myocyte fractional shortening from 3.8+/-0.6 to 9.6+/-0.8% and prolonged action potential duration at 30% repolarisation from 9+/-2 to 102+/-11 ms. Clamping cells to an AP waveform recorded during exposure to AVE0118 produced the same inotropic response as the drug itself. In action potential clamp, peak Ca2+ inward current (I(CaL)) current declined from 5.5+/-1.3 pA/pF during control to 4.1+/-0.7 pA/pF when an AP recorded in the presence of AVE0118 was used as command waveform. However, I(CaL) was more sustained with AVE0118 and the time integral did not change (135+/-37 vs. 173+/-30 pA/pFms, p = ns). Importantly, blockade of reverse mode Na+/Ca2+-exchanger activity with 5 microM KBR7943 or using a Na+-free pipette solution abolished the positive inotropic effect of the AP recorded in the presence of AVE0118. In ventricular myocytes AVE0118 did not elicit a positive inotropic response. CONCLUSIONS: Block of I(Kur) by AVE0118 enhances atrial contractility both in patients with sinus rhythm and atrial fibrillation. The positive inotropic effect is atrial-specific and due to the changes of the action potential configuration which enhances Ca2+ entry via reverse mode Na+/Ca2+ exchange.

AVE0118, blocker of the transient outward current (I(to)) and ultrarapid delayed rectifier current (I(Kur)), fully restores atrial contractility after cardioversion of atrial fibrillation in the goat.

BACKGROUND: The loss of atrial contractile function after cardioversion of atrial fibrillation (AF) contributes to the thromboembolic risk associated with AF. The newly developed blocker of the transient outward current (I(to)) and ultrarapid delayed rectifier current (I(Kur)) AVE0118 prolongs atrial action potential duration and might therefore enhance atrial contractility. We compared the ability of AVE0118 to restore atrial contraction after cardioversion of AF with the efficacy of conventional positive inotropic compounds in the goat model of AF. METHODS AND RESULTS: Eighteen goats were chronically instrumented with epicardial electrodes, a pressure transducer in the right atrium, and piezoelectric crystals to measure right atrial diameter. Atrial contractility and refractoriness and QT duration were measured before and after 1 week (3 to 8 days) of AF induced by repetitive burst pacing. The measurements were repeated after administration of digoxin (0.02 mg/kg), dobutamine (5 microg x kg(-1) x min(-1)), the Ca2+ sensitizer EMD57033 (1 mg x kg(-1) x min(-1)), the L-type Ca2+ channel agonist BayY5959 (0.1 mg x kg(-1) x min(-1)), and AVE0118 (0.01 to 0.2 mg x kg(-1) x min(-1)). The effect of AVE0118 on the configuration of atrial monophasic action potentials was determined for comparison. After 1 week of AF, atrial contractility during sinus rhythm or slow atrial pacing was reduced to <10%. Digoxin and dobutamine failed to increase atrial contractility. EMD57033 restored 41% and BayY5959 restored 48% of atrial contractility at baseline. BayY5959 significantly prolonged QT duration by 24.7%. AVE0118 enhanced atrial contraction to 156% of the baseline value. The positive inotropic effect was accompanied by a pronounced prolongation of atrial action potential duration and refractoriness, whereas QT duration remained unchanged. CONCLUSIONS: Conventional positive inotropic drugs showed limited effect on atrial contractility after cardioversion of AF or produced QT prolongation. In contrast, the I(to)/I(Kur) blocker AVE0118 fully restored atrial contraction without proarrhythmic effects on the ventricle.

Prostaglandin F2 alpha induces unsynchronized intracellular calcium oscillations in monolayers of gap junctionally coupled NRK fibroblasts.

We investigated the intracellular calcium oscillations induced by prostaglandin F2alpha (PGF2alpha) in individual cells of confluent, gap junction-coupled monolayers of normal rat kidney (NRK) fibroblasts. PGF2alpha (1000 nM) induced oscillations in more than 90% of the cells in the monolayer, but the frequency of these oscillations was highly variable between individual cells (0.2-1.4 min(-1)). The initial calcium peak resulted from calcium release from IP3-sensitive stores, while subsequent calcium transients were mediated by interplay between both IP3-sensitive calcium stores and calcium influx. The oscillation frequency was increased by sensitizing the IP3 receptor with thimerosal (10 microM) and depended on the extracellular calcium concentration. Thapsigargin (5 nM), which inhibits reuptake of calcium into the stores, only seemed to reduce the amplitude of the oscillation. Patch-clamp experiments revealed that PGF2alpha did not inhibit electrical coupling of the NRK cells in the monolayer. Gap junctional permeability of NRK cells thus appears to be sufficient to allow electrical coupling, resulting in a uniform membrane potential throughout the entire monolayer, but insufficient to synchronize the intracellular calcium oscillations upon PGF2alpha stimulation.

Besides affecting intracellular calcium signaling, 2-APB reversibly blocks gap junctional coupling in confluent monolayers, thereby allowing measurement of single-cell membrane currents in undissociated cells.

2-aminoethoxydiphenyl borate (2-APB) has been widely used as a blocker of the IP3 receptor and TRP channels, including store-operated calcium channels. We now show in monolayers of normal rat kidney cells (NRK/49F) that 2-APB completely and reversibly blocks gap junctional intercellular communication at concentrations similar to that required for inhibition of PGF2alpha-induced increases in intracellular calcium. Gap junctional conductances between NRK cells were estimated with single-electrode patch-clamp measurements and were fully blocked by 2-APB (50 microM), when applied extracellularly but not via the patch pipette. Half maximal inhibition (IC50) of electrical coupling in NRK cells was achieved at 5.7 microM. Similar results were obtained for human embryonic kidney epithelial cells (HEK293/tsA201) with an IC50 of 10.3 microM. Using 2-APB as an electrical uncoupler of monolayer cells, we could thus measure inward rectifier potassium, L-type calcium, and calcium-dependent chloride membrane currents in confluent NRK monolayers, with properties similar to those in dissociated NRK cells in the absence of 2-APB. The electrical uncoupling action described here is a new 2-APB property that promises to provide a powerful pharmacological tool to study single-cell properties in cultured confluent monolayers and intact tissues by electrical and chemical uncoupling of the cells without the need of prior dissociation.

Lipid factor (bVLF) from bovine vitreous body evokes in EGFR-T17 cells a Ca2+-dependent K+ current associated with inositol 1,4,5-trisphosphate-independent Ca2+ mobilization.

Bovine vitreous lipid factor (bVLF) is a complex phospholipid isolated from bovine vitreous body with strong Ca(2+)-mobilizing activity. In this study, the effects of bVLF on membrane potential were investigated in EGFR-T17 fibroblasts with the whole-cell patch clamp technique on monolayer cells, as well as with the fluorescent dye bis-oxonol as membrane potential-sensitive probe on monolayer and suspension cells. bVLF induced a transient hyperpolarization characterized by an initial peak and subsequent return to resting membrane potential levels within 1-2 min. The increase of [Ca(2+)](i) was concomitant with an outward current responsible for the hyperpolarizing response. Results with: (a) high [K(+)](o) media; (b) the monovalent cation ionophore gramicidin; and (c) substitution of K(+) with Cs(+) in the intracellular solution were consistent with the involvement of K(+) channels. The bVLF-induced hyperpolarization was blocked by the K(+) channel blockers, quinine and tetraethylamonium chloride, and partially affected by 4-aminopyridine. The calcium ionophore ionomycin caused a similar hyperpolarization as bVLF. When intracellular calcium was buffered by adding BAPTA to the pipette solution, bVLF-activated outward current was prevented. Moreover, the hyperpolarization response was strongly reduced at low doses (3 nM) of specific Ca(2+)-activated K(+) channel blockers, charybdotoxin and iberiotoxin. Based on these observations we conclude that bVLF hyperpolarizes the cells via the activation of a Ca(2+)-dependent K(+) current. In addition, it was observed that bVLF did not have a significant effect on intercellular communication measured by a single patch-electrode technique. Thus, membrane potential changes appeared to belong to the earliest cellular responses triggered by bVLF, and are closely associated with phosphatidic acid-dependent [Ca(2+)](i) mobilization.