Optical Action Potentials Research Articles

LKB1 is a Critical Regulator of Early Atrial Growth and Electrophysiological Function

The liver kinase B1 (LKB1)-AMP activated protein kinase (AMPK) pathway regulates cellular metabolism, polarity, and growth. Although previous studies have documented the maladaptive consequences of LKB1 deletion in the progressive development of atrial fibrillation (AF) and cardiac hypertrophy, its role in chamber-specific pathophysiology remains largely unresolved. Therefore we systematically explored LKB1's differential effects on structural and ion channel remodeling in mice with LKB1 deletion in cardiomyocytes. Atrial but not ventricular Nav1.5 and Cx40 expression levels were markedly downregulated in 1d neonates, giving rise to a 2-fold prolongation of the P wave on surface ECG. Detailed ex vivo optical action potential (AP) mapping studies revealed presence of marked inter-atrial conduction abnormalities, loss of bi-atrial electrical coupling, and prolonged AP durations in the atria of mice with LKB1 deletion. In contrast, ventricular changes developed with a much slower time-course that was consistent with previous studies. AMPK α2 inactivated hearts demonstrated modest overlap in ion channel expression, but retained normal ECG characteristics, cardiac structure and function compared to their LKB1 deleted counterparts. LKB1 deletion triggers a primary atrial electrophysiological remodeling program that precedes structural abnormalities and likely underlies the development of AF. LKB1 may be a novel target for the prevention of AF.

Nicorandil improves electrical remodelling, leading to the prevention of electrically induced ventricular tachyarrhythmia in a mouse model of desmin-related cardiomyopathy

1. Transgenic (TG) mice overexpressing an arg120gly missense mutation in heat shock protein B5 (HSPB5; i.e. R120G TG mice) exhibit desmin-related cardiomyopathy. Recently, the cardioprotective effect of nicorandil has been shown to prolong the survival of R120G TG mice. However, whether the TG mice exhibit ventricular arrhythmias and whether nicorandil can inhibit these arrhythmias remain unknown. In the present study we examined the effects of chronic nicorandil administration on ventricular electrical remodelling and arrhythmias in R120G TG mice. 2. Mice were administered nicorandil (15 mg/kg per day) or vehicle (water) orally from 5 to 30 weeks of age. Electrocardiograms (ECG) and optical action potentials were recorded from R120G TG mouse hearts. In addition, the expression of ventricular connexin 43 and the cardiac Na(+) channel Nav1.5 was examined in TG mice. 3. All ECG parameters tested were prolonged in R120G TG compared with non-transgenic (NTG) mice. Nicorandil improved the prolonged P, PQ and QRS intervals in R120G TG mice. Interestingly, impulse conduction slowing and increases in the expression of total and phosphorylated connexin 43 and Nav1.5 were observed in ventricles from R120G TG compared with NTG mice. Nicorandil improved ventricular impulse conduction slowing and normalized the increased protein expression levels of total and phosphorylated connexin 43, but not of Nav1.5, in R120G TG mouse hearts. Electrical rapid pacing at the ventricle induced ventricular tachyarrhythmias (VT) in six of eight R120G TG mouse hearts, but not in any of the eight nicorandil-treated R120G TG mouse hearts (P < 0.05). 4. These findings demonstrate that nicorandil inhibits cardiac electrical remodelling and that the prevention of VT by nicorandil is associated with normalization of connexin 43 expression in this model.

Transmural Ultrasound Imaging of Thermal Lesion and Action Potential Changes in Perfused Canine Cardiac Wedge Preparations by High Intensity Focused Ultrasound Ablation

Intra-procedural imaging is important for guiding cardiac arrhythmia ablation. It is difficult to obtain intra-procedural correlation of thermal lesion formation with action potential (AP) changes in the transmural plane during ablation. This study tested parametric ultrasound imaging for transmural imaging of lesion and AP changes in high intensity focused ultrasound (HIFU) ablation using coronary perfused canine ventricular wedge preparations (n = 13). The preparations were paced from epi/endocardial surfaces and subjected to HIFU application (3.5 MHz, 11 Hz pulse-repetition-frequency, 70% duty cycle, duration 4 s, 3500 W/cm2), during which simultaneous optical mapping (1 kframes/s) using di-4-ANEPPS and ultrasound imaging (30 MHz) of the same transmural surface of the wedge were performed. Spatiotemporally correlated AP measurements and ultrasound imaging allowed quantification of the reduction of AP amplitude (APA), shortening of AP duration at 50% repolarization, AP triangulation, decrease of optical AP rise, and change of conduction velocity along tissue depth direction within and surrounding HIFU lesions. The threshold of irreversible change in APA correlating to lesions was determined to be 43±1% with a receiver operating characteristic (ROC) area under curve (AUC) of 0.96±0.01 (n = 13). Ultrasound imaging parameters such as integrated backscatter, Rayleigh (α) and log-normal (σ) parameters, cumulative extrema of σ were tested, with the cumulative extrema of σ performing the best in detecting lesion (ROC AUC 0.89±0.01, n = 13) and change of APA (ROC AUC 0.79±0.03, n = 13). In conclusion, characteristic tissue and AP changes in HIFU ablation were identified and spatiotemporally correlated using optical mapping and ultrasound imaging. Parametric ultrasound imaging using cumulative extrema of σ can detect HIFU lesion and APA reduction.

Abstract 217: Effect of Amiodarone with Therapeutic Hypothermia on Arrhythmia Substrates During Global Myocardial Ischemia

Background: The effect of therapeutic hypothermia (TH) after cardiac arrest on susceptibility to arrhythmias is not well understood. We have shown that TH has a beneficial effect on reentrant substrates for ischemia-induced arrhythmias by limiting decreases in conduction velocity (CV) and attenuating increases in transmural dispersion of repolarization (DOR). As amiodarone is utilized during TH, it is important to understand the effect of TH and amiodarone on arrhythmia substrates during ischemia. Our hypothesis is that amiodarone worsens the antiarrhythmic effect of TH during global ischemia. To test this hypothesis, a model of no flow global ischemia in the canine wedge preparation was used to study the effects of ischemia on all cell types spanning the transmural wall. Methods: Optical action potentials were recorded transmurally with high spatial (1 mm), temporal (.5 ms) and voltage (.5mv) resolution. Amiodarone (5microM) was used at both 36oC (control, CTAmio n=4) and 32oC (THAmio, n=4) during 15 min of no flow global ischemia and compared to controls (CT=7, TH=6). Action potential duration (APD), DOR, and CV were measured for all conditions. Programmed electrical stimulation (PES) was performed to assess for ischemia-induced arrhythmias and compared to prior controls (CT, n=5, TH, n=4). Results: At baseline, TH prolonged APD in both THAmio and TH, but there was no difference between DOR and CV. At 15 minutes of ischemia, DOR increased in the THAmio group compared to TH (34±3 ms to 79±9 ms vs. 25±3 to 37±7 ms, p=.04). There was a trend towards worsening ischemia-induced conduction slowing in the THAmio group vs TH, but not significant (39±4 to 24±3 cm/s vs 44±2 to 35±3 cm/s, p=.06). There was no difference under control temperature of CTAmio vs CT for CV, DOR, or APD. PES induced arrhythmias in 2/4 in the CTAmio group vs. 0/4 in THAmio group (compared to 3/5 in CT group, 1/4 in TH group). Conclusions: Amiodarone worsens the beneficial effect of TH on ischemia-induced DOR, but does not worsen susceptibility to arrhythmias. Further study in translatable models of ischemia-induced cardiac arrest is warranted to assess the effect of ACLS pharmacology on arrhythmias with the increased use of TH.

Abstract 211: Blockade of Na+-Sensitive K+ Channels Alleviates Frequency-Dependent Electrical Failure in an Ex-Vivo Model of VF-Induced Cardiac Arrest

Ventricular fibrillation (VF), global ischemia and beta-adrenergic stimulation form the common context of sudden cardiac arrest. Regional and global loss of excitability under these conditions sets the stage of resuscitation failure due to either asystole or post-reperfusion VF recurrence. We hypothesized that Na+-sensitive K+ current (IKNa) significantly contributes to electrical failure under combined conditions of ischemia, high excitation frequency, and beta-adrenergic stimulation. We performed optical mapping of voltage-sensitive dye RH237 in the anterior right and left ventricle (RV and LV) of Langendorff-perfused rabbit hearts subjected to normothermic global no-flow ischemia, isoproterenol (30 nM) and pacing at the cycle length (CL) of either 300 ms or 200 ms. 2,3-butanedione monoxime (20 mM) was used to reduce motion artifact. Excitable area was measured in optical maps based on the criterion of the minimum time-derivative of the upstroke of the optical action potential. Decrease of the pacing CL from 300 to 200 ms dramatically accelerated loss of excitability in both RV and LV (Figure, p&lt;0.0001). Treatment with IKNa blocker R56865 significantly postponed frequency-dependent electrical failure (p&lt;0.0001), the protective effect being larger in RV than in LV (see Figure). After 5 min of reperfusion, R56865 treatment group also showed better recovery of excitability, effect being predominant in the LV in comparison to 200 ms CL group. This is the first study to show that IKNa activation can promote electrical failure under conditions mimicking VF-induced cardiac arrest.

Abstract 201: Beta-Adrenergic Stimulation Promotes Early Electrical Failure During Simulated VF-Induced Cardiac Arrest And Delays Electrical Recovery Upon Reperfusion

During cardiac arrest adrenergic stimulation is enhanced due to both endogenous increase in myocardial tissue catecholamine levels and exogenous epinephrine administration during resuscitation. Whereas alpha-adrenergic stimulation may be beneficial for hemodynamic recovery, there is growing evidence that beta-adrenergic stimulation is detrimental. Clinically, there is controversy as to whether epinephrine administration improves overall survival in cardiac arrest. Here we investigated the effects of beta-adrenergic stimulation on electrical activity during simulated ventricular fibrillation (VF) [[Unable to Display Character: –]] induced cardiac arrest and subsequent reperfusion. We performed optical mapping of the voltage-sensitive dye RH237 in the anterior right and left ventricles (RV and LV) of Langendorff-perfused rabbit hearts subjected to normothermic global no-flow ischemia and rapid pacing (cycle length 200 ms) in the presence or in the absence of isoproterenol (ISO, 30 nM). 2,3-butanedione monoxime (20 mM) was used to reduce motion artifact. Excitable area was measured in optical maps based on the criterion of the minimum time-derivative of the upstroke of the optical action potential. ISO markedly accelerated electrical depression in both LV and RV (more in LV) compared to control hearts (see Figure, * indicate comparisons with significant difference between groups at p&lt;0.05). In addition, ISO delayed recovery of electrical activity upon reperfusion (62±14% recovery in ISO vs.98±2% recovery in control at 5 min of reperfusion, p&lt;0.01). Furthermore, ISO tended to increase the incidence of post-reperfusion arrhythmias compared to control (83% vs. 25%, respectively; p=NS). We conclude that beta-adrenergic stimulation aggravates electrical dysfunction under conditions typically present during cardiac arrest/resuscitation, which might contribute to the overall detrimental outcomes of natural or induced catecholamine increase in this setting.

In situ investigation of allografted mouse HCN4 gene–transfected rat bone marrow mesenchymal stromal cells with the use of patch-clamp recording of ventricular slices

BackgroundRecently, proof-of-concept experiments have shown that genetically modified bone marrow mesenchymal stromal cells (MSCs) carrying hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were able to express the funny current (If) in vitro, which played a key role in the process of pacemaker generation for heart rate, and were capable of pacemaker function after transplantation into the host heart. Nevertheless, because of the lack of direct experimental access to the implanted cells in situ, the changes in electrophysiological characteristics and the mechanisms underlying the pacemaker function of engrafted HCN gene–transfected MSCs in vivo remain unclear. Methods and ResultsOn the basis of the improved preparation of ventricular slices, we successfully performed an in situ investigation of allografted mouse HCN4 gene (mHCN4)-transfected rat MSCs (rMSCs) with the use of patch-clamp recording in ventricular slices. We demonstrate that allografted mHCN4-transfected rMSCs survived in the host heart for >4 weeks; that they expressed If, which is generated by the mHCN4 channel, with a similar amplitude but greater negative activation compared with parallel cells cultured in vitro; that they did not express optical action potentials or depolarization-activated inward sodium or calcium currents; and that they exhibited a low incidence of gap-junctional coupling with host cardiomyocytes. ConclusionsThis study provides direct experimental access to investigate MSCs after transplantation into the host heart. We propose that mHCN4-transfected rMSCs survived in the host heart with altered electrophysiological characteristics of If and were accompanied by a low efficiency of connexin 43 expression at 4 weeks after transplantation, which may affect its pacemaker function in vivo.

The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart

Blebbistatin (BS) is a recently discovered inhibitor of the myosin II isoform and has been adopted as the mechanical uncoupler of choice for optical mapping, because previous studies suggest that BS has no significant cardiac electrophysiological effects in a number of species. The aim of this study was to determine whether BS affects cardiac electrophysiology in isolated New Zealand White rabbit hearts. Langendorff-perfused hearts (n= 39) in constant-flow mode had left ventricular monophasic action potential duration (MAPD) measured at apical and basal regions during constant pacing (300 ms cycle length). Standard action potential duration restitution was obtained using the single extrastimulus method with measurement of the maximal restitution slope. Ventricular fibrillation threshold was measured as the minimal current inducing sustained ventricular fibrillation with burst pacing (30 stimuli, at 30 ms intervals). Optical action potentials were recorded using the voltage-sensitive dye di-4-ANEPPS. Measurements were taken at baseline and after 60 min perfusion with BS (5 μm). Blebbistatin significantly prolonged left ventricular apical (mean ± SEM; from 129.9 ± 2.9 to 170.7 ± 4.1 ms, P < 0.001, n= 8) and basal MAPD (from 135.0 ± 2.3 to 163.3 ± 5.6 ms, P < 0.001) and effective refractory period (from 141.3 ± 4.8 to 175.6 ± 3.7 ms, P < 0.001) whilst increasing the maximal slope of restitution (apex, from 0.79 ± 0.09 to 1.57 ± 0.16, P < 0.001; and base, from 0.71 ± 0.06 to 1.44 ± 0.24, P < 0.001) and ventricular fibrillation threshold (from 5.3 ± 1.1 to 17.0 ± 2.9 mA, P < 0.001). In other hearts, blebbistatin significantly prolonged optically recorded action potentials (from 136.5 ± 6.3 to 173.0 ± 7.9 ms, P < 0.05, n= 4). In control experiments, the increase of MAPD with blebbistatin was present whether the hearts were perfused in constant-pressure mode (n= 5) or in unloaded conditions (n= 5). These data show that blebbistatin significantly affects cardiac electrophysiology. Its use in optical mapping studies should be treated with caution.

209 EMF Preservation of Gap Junction Coupling Improves Transmural Conduction Velocity and Limits Epicardial Conduction Block During Global Myocardial Ischemia

Study Objectives: Hypothermia and myocardial ischemia are both arrhythmogenic by several mechanisms, including transmural conduction slowing and increased dispersion of repolarization. However, therapeutic hypothermia has not been associated with increased arrhythmias after return of spontaneous circulation after cardiac arrest. Preliminary work in our lab suggests that therapeutic hypothermia may be antiarrhythmic by attenuating transmural slowing of ventricular conduction velocity, limiting epicardial conduction block, and attenuating ischemia-induced dispersion of repolarization. Preserving intracellular gap junction coupling has been shown to improve conduction during ischemia. Improving gap junction coupling may be a potential mechanism for the improved conduction velocity and decrease in epicardial conduction block observed with mild hypothermia in our canine model of global ischemia. Our aim was to investigate the effect of rotagaptide, a gap junction opener, on the transmural electrophysiologic effects of global ischemia in ventricular myocardium.Methods: Langendorff-perfused canine left ventricle wedge preparations were pre-treated with the gap junction opener rotigaptide (R) (50 nM, n=8) compared to historic control group (C) (n=7) at normal temp (36°C). Optical action potentials were recorded with high spatial (1 mm), temporal (.5 ms) and voltage (.5mv) resolution from cells spanning the transmural wall of the left ventricle. Wedges underwent global no-flow ischemia for 15 min and subsequent reperfusion. Action potential duration, conduction velocity, dispersion of repolarization, and evidence of epicardial conduction block were recorded.Results: No difference in conduction velocity or mean APD was seen at baseline between the 2 groups. At 15 min ischemia, rotigaptide attenuated the ischemia induced conduction slowing, with a conduction velocity decrease of 31±8% versus 50±13% (p=.02) Mean APD was preserved in the R group (222±4 ms to 221±12 ms) versus APD shortening in the C group (230±6 ms to 178±13 ms, p=.01), and the severe epicardial APD shortening seen in the C group from baseline to 15 min of ischemia (211±7 ms to 142±15ms, p=.004) was attenuated in R group (209±5 ms to 193±20ms, p=NS). Epicardial conduction block was observed in only 2/8 preparations in the R group versus 6/7 in the C group. Conduction velocity and Mean APD returned to baseline upon reperfusion.Conclusion: Preservation of gap junction coupling attenuated ischemia-induced slowing of transmural conduction velocity, preserved mean APD, and limited ischemia-induced epicardial conduction block, and suggests a mechanism for the similar effect seen in therapeutic hypothermia. Study Objectives: Hypothermia and myocardial ischemia are both arrhythmogenic by several mechanisms, including transmural conduction slowing and increased dispersion of repolarization. However, therapeutic hypothermia has not been associated with increased arrhythmias after return of spontaneous circulation after cardiac arrest. Preliminary work in our lab suggests that therapeutic hypothermia may be antiarrhythmic by attenuating transmural slowing of ventricular conduction velocity, limiting epicardial conduction block, and attenuating ischemia-induced dispersion of repolarization. Preserving intracellular gap junction coupling has been shown to improve conduction during ischemia. Improving gap junction coupling may be a potential mechanism for the improved conduction velocity and decrease in epicardial conduction block observed with mild hypothermia in our canine model of global ischemia. Our aim was to investigate the effect of rotagaptide, a gap junction opener, on the transmural electrophysiologic effects of global ischemia in ventricular myocardium. Methods: Langendorff-perfused canine left ventricle wedge preparations were pre-treated with the gap junction opener rotigaptide (R) (50 nM, n=8) compared to historic control group (C) (n=7) at normal temp (36°C). Optical action potentials were recorded with high spatial (1 mm), temporal (.5 ms) and voltage (.5mv) resolution from cells spanning the transmural wall of the left ventricle. Wedges underwent global no-flow ischemia for 15 min and subsequent reperfusion. Action potential duration, conduction velocity, dispersion of repolarization, and evidence of epicardial conduction block were recorded. Results: No difference in conduction velocity or mean APD was seen at baseline between the 2 groups. At 15 min ischemia, rotigaptide attenuated the ischemia induced conduction slowing, with a conduction velocity decrease of 31±8% versus 50±13% (p=.02) Mean APD was preserved in the R group (222±4 ms to 221±12 ms) versus APD shortening in the C group (230±6 ms to 178±13 ms, p=.01), and the severe epicardial APD shortening seen in the C group from baseline to 15 min of ischemia (211±7 ms to 142±15ms, p=.004) was attenuated in R group (209±5 ms to 193±20ms, p=NS). Epicardial conduction block was observed in only 2/8 preparations in the R group versus 6/7 in the C group. Conduction velocity and Mean APD returned to baseline upon reperfusion. Conclusion: Preservation of gap junction coupling attenuated ischemia-induced slowing of transmural conduction velocity, preserved mean APD, and limited ischemia-induced epicardial conduction block, and suggests a mechanism for the similar effect seen in therapeutic hypothermia.

The quest for rotors in atrial fibrillation: Different nets catch different fishes

The quest for rotors in atrial fibrillation: Different nets catch different fishes

Use of endogenous NADH fluorescence for real-time in situ visualization of epicardial radiofrequency ablation lesions and gaps

Radiofrequency ablation (RFA) aims to produce lesions that interrupt reentrant circuits or block the spread of electrical activation from sites of abnormal activity. Today, there are limited means for real-time visualization of cardiac muscle tissue injury during RFA procedures. We hypothesized that the fluorescence of endogenous NADH could be used as a marker of cardiac muscle injury during epicardial RFA procedures. Studies were conducted in blood-free and blood-perfused hearts from healthy adult Sprague-Dawley rats and New Zealand rabbits. Radiofrequency was applied to the epicardial surface of the heart using a 4-mm standard blazer ablation catheter. A dual camera optical mapping system was used to monitor NADH fluorescence upon ultraviolet illumination of the epicardial surface and to record optical action potentials using the voltage-sensitive probe RH237. Epicardial lesions were seen as areas of low NADH fluorescence. The lesions appeared immediately after ablation and remained stable for several hours. Real-time monitoring of NADH fluorescence allowed visualization of viable tissue between the RFA lesions. Dual recordings of NADH and epicardial electrical activity linked the gaps between lesions to postablation reentries. We found that the fluorescence of endogenous NADH aids the visualization of injured epicardial tissue caused by RFA. This was true for both blood-free and blood-perfused preparations. Gaps between NADH-negative regions revealed unablated tissue, which may promote postablation reentry or provide pathways for the conduction of abnormal electrical activity.

Amplitude Changes during Ventricular Fibrillation: A Mechanistic Insight

Introduction: Clinically in ventricular fibrillation (VF), ECG amplitude, and frequency decrease as ischemia progresses and predict defibrillation success. In vitro ECG amplitude declines without ischemia, independent of VF frequencies. This study examines the contribution of cellular electrical activity and global organization to ECG amplitude changes during VF. Methods and Results: Rabbit hearts were Langendorff-perfused (40 mL/min, Tyrode’s solution) and loaded with RH237. During VF, ECG, and epicardial optical action potentials were recorded (photodiode array; 256 sites, 15 mm × 15 mm). After 60 s of VF, perfusion was either maintained, global ischemia produced by low-flow (6 mL/min), or solution [K+]o raised to 8 mM. Peak-to-peak amplitude was determined for all signals. During VF, in control, ECG amplitude decreased to a steady-state (∼57% baseline), whereas in low-flow steady-state was not reached with the amplitude continuing to fall to 33% of baseline by 600 s. Optically, LV amplitude declined more than RV, reaching significance in control (LV vs. RV; 33 ± 5 vs. 63 ± 8%, p < 0.01). During VF in 8 mM [K+]o, amplitude changes were more complex; ECG amplitude increased with time (105 ± 13%), whilst LV amplitude decreased (60 ± 15%, p < 0.001). Microelectrode studies showed amplitude reduction in control and 8 mM [K+]o (to ∼79 and ∼93% baseline, respectively). Evaluation of electrical coordination by cross-correlation of optical signals showed as VF progressed coordination reduced in control (baseline 0.36 ± 0.02 to 0.28 ± 0.003, p < 0.01), maintained in low-flow (0.41 ± 0.03 to 0.37 ± 0.005, p = NS) and increased in 8 mM [K+]o (0.36 ± 0.02 to 0.53 ± 0.08, p < 0.05). Conclusion: ECG amplitude decline in VF is due to a combination of decreased systolic activation at the cellular level and increased desynchronization of inter-cellular electrical activity.

Construction of 3D MR image-based computer models of pathologic hearts, augmented with histology and optical fluorescence imaging to characterize action potential propagation

Cardiac computer models can help us understand and predict the propagation of excitation waves (i.e., action potential, AP) in healthy and pathologic hearts. Our broad aim is to develop accurate 3D MR image-based computer models of electrophysiology in large hearts (translatable to clinical applications) and to validate them experimentally. The specific goals of this paper were to match models with maps of the propagation of optical AP on the epicardial surface using large porcine hearts with scars, estimating several parameters relevant to macroscopic reaction-diffusion electrophysiological models. We used voltage-sensitive dyes to image AP in large porcine hearts with scars (three specimens had chronic myocardial infarct, and three had radiofrequency RF acute scars). We first analyzed the main AP waves' characteristics: duration (APD) and propagation under controlled pacing locations and frequencies as recorded from 2D optical images. We further built 3D MR image-based computer models that have information derived from the optical measures, as well as morphologic MRI data (i.e., myocardial anatomy, fiber directions and scar definition). The scar morphology from MR images was validated against corresponding whole-mount histology. We also compared the measured 3D isochronal maps of depolarization to simulated isochrones (the latter replicating precisely the experimental conditions), performing model customization and 3D volumetric adjustments of the local conductivity. Our results demonstrated that mean APD in the border zone (BZ) of the infarct scars was reduced by ~13% (compared to ~318 ms measured in normal zone, NZ), but APD did not change significantly in the thin BZ of the ablation scars. A generic value for velocity ratio (1:2.7) in healthy myocardial tissue was derived from measured values of transverse and longitudinal conduction velocities relative to fibers direction (22 cm/s and 60 cm/s, respectively). The model customization and 3D volumetric adjustment reduced the differences between measurements and simulations; for example, from one pacing location, the adjustment reduced the absolute error in local depolarization times by a factor of 5 (i.e., from 58 ms to 11 ms) in the infarcted heart, and by a factor of 6 (i.e., from 60 ms to 9 ms) in the heart with the RF scar. Moreover, the sensitivity of adjusted conductivity maps to different pacing locations was tested, and the errors in activation times were found to be of approximately 10-12 ms independent of pacing location used to adjust model parameters, suggesting that any location can be used for model predictions.

Abstract 256: Therapeutic Hypothermia Decreases Arrhythmia Substrates by Attenuating Repolarization Current I K,ATP During Myocardial Ischemia

Background: While hypothermia is arrhythmogenic, the effect of therapeutic hypothermia (TH) performed after cardiac arrest on susceptibility to arrhythmias is not well understood. Clinically, patients do not have an increase in arrhythmias, and it is possible that TH may be cardioprotective. The repolarization current I K,ATP is activated only during ischemia where it heterogeneously shortens action potentials increasing dispersion of repolarization (DOR). Since I K,ATP is attenuated at cooler temperatures, we hypothesized that the antiarrhythmic effect of TH during ischemia may be mediated by attenuation of I K,ATP activation. To test this hypothesis, a model of no flow global ischemia in the canine wedge preparation was created to study the effects of ischemia on all cell types spanning the transmural wall. Methods: Optical action potentials were recorded transmurally with high spatial (1 mm), temporal (.5 ms) and voltage (.5mv) resolution. To block I K,ATP, glibenclamide (10 µmol) was used at both 36 o C (normal temperature, NT+ I K,ATP block, n=3) and 32 o C (TH+ I K,ATP block, n=3) during 15 min of no flow global ischemia. I K,ATP blockade was compared to prior controls (NT=7, TH=6). Action potential duration (APD) for epicardial, mid-myocardial, and epicardial cells, DOR, and conduction velocity (CV) were measured for all conditions. Results: There was no difference in DOR between the 4 groups prior to ischemia. NT+ I K,ATP block attenuated ischemia-induced increase in DOR compared to NT controls (22±4 ms to 25±5 ms vs. 30±5 to 57±5 ms, p=.002). NT+ I K,ATP block also attenuated ischemia-induced conduction slowing (44±2 to 30±3 cm/s vs 34±5 to 18±3 cm/s, p=.03). Addition of I K,ATP block to TH did not further attenuate the DOR or CV vs. TH alone (p&gt;0.05). Ischemia-induced epicardial conduction bock was attenuated by I K,ATP blockade, (0/3 I K,ATP block vs. 6/7 NT) Epicardial block was attenuated in both TH groups. Conclusions: I K,ATP blockade attenuates ischemia-induced DOR, conduction slowing, and epicardial conduction block, reproducing the effect of TH. This suggests that hypothermia-induced attenuation of I K,ATP may play a role in the mechanism of attenuation of ischemia-induced arrhythmias observed in TH.

Optical mapping of cryoinjured rat myocardium grafted with mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been shown to improve cardiac electrophysiology when administered in the setting of acute myocardial infarction. However, the electrophysiological phenotype of MSCs in situ is not clear. We hypothesize that MSCs delivered intramyocardially to cryoinjured myocardium can engraft, but will not actively generate, action potentials. Cryoinjury-induced scar was created on the left ventricular epicardial surface of adult rat hearts. Within 30 min, hearts were injected with saline (sham, n = 11) or bone marrow-derived MSCs (2 × 10(6)) labeled with 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine percholate (DiI; n = 16). At 3 wk, optical mapping and cell isolation were used to measure optical action potentials and calcium transients, respectively. Histological analysis confirmed subepicardial scar thickness and the presence of DiI-positive cells that express connexin-43. Optical action potential amplitude within the scar at MSC-positive sites (53.8 ± 14.3%) was larger compared with sites devoid of MSCs (35.3 ± 14.2%, P < 0.05) and sites within the scar of shams (33.5 ± 6.9%, P < 0.05). Evidence of simultaneous action potential upstroke, the loss of action potential activity following ablation of adjacent viable myocardium, and no rapid calcium transient response in isolated DiI+ cells suggest that the electrophysiological influence of engrafted MSCs is electrotonic. MSCs can engraft when directly injected into a cryoinjury and are associated with evidence of action potential activity. However, our results suggest that this activity is not due to generation of action potentials, but rather passive influence coupled from neighboring viable myocardium.

Anatomic Localization and Autonomic Modulation of Atrioventricular Junctional Rhythm in Failing Human Hearts

The structure-function relationship in the atrioventricular junction (AVJ) of various animal species has been investigated in detail; however, less is known about the human AVJ. In this study, we performed high-resolution optical mapping of the human AVJ (n = 6) to define its pacemaker properties and response to autonomic stimulation. Isolated, coronary-perfused AVJ preparations from failing human hearts (n = 6, 53 ± 6 years) were optically mapped using the near-infrared, voltage-sensitive dye, di-4-ANBDQBS, with isoproterenol (1 μmol/L) and acetylcholine (1 μmol/L). An algorithm detecting multiple components of optical action potentials was used to reconstruct multilayered intramural AVJ activation and to identify specialized slow and fast conduction pathways (SP and FP). The anatomic origin and propagation of pacemaker activity was verified by histology. Spontaneous AVJ rhythms of 29 ± 11 bpm (n = 6) originated in the nodal-His region (n = 3) and/or the proximal His bundle (n = 4). Isoproterenol accelerated the AVJ rhythm to 69 ± 12 bpm (n = 5); shifted the leading pacemaker to the transitional cell regions near the FP and SP (n = 4) and/or coronary sinus (n = 2); and triggered reentrant arrhythmias (n = 2). Acetylcholine (n = 4) decreased the AVJ rhythm to 18 ± 4 bpm; slowed FP/SP conduction leading to block between the AVJ and atrium; and shifted the pacemaker to either the transitional cell region or the nodal-His region (bifocal activation). We have demonstrated that the AVJ pacemaker in failing human hearts is located in the nodal-His region or His bundle regions and can be modified with autonomic stimulation. Moreover, we found that both the FP and SP are involved in anterograde and retrograde conduction.

Deciphering Arrhythmia Mechanisms: Tools of the Trade

Pathophysiological remodeling of cardiac function occurs at multiple levels, spanning the spectrum from molecular and sub-cellular changes to those occurring at the organ-system levels. Of key importance to arrhythmias are changes in electrophysiological and calcium handling properties at the tissue level. In this review, we discuss how high-resolution optical action potential and calcium transient imaging has advanced our understanding of basic arrhythmia mechanisms associated with multiple cardiovascular disorders, including the long QT syndrome, heart failure, and ischemia-reperfusion injury. We focus on the role of repolarization gradients (section 1) and calcium mediated triggers (section 2) in the initiation and maintenance of complex arrhythmias in these settings.

Photon Diffusion Attenuation Length in Tyrode and Blood-Perfused Myocardial Tissue

Photon attenuation length (δ) in biological tissues determines interrogation depth, spatial resolution, and amplitude of fluorescence signal in various types of optical imaging, including imaging of cardiac excitation using voltage-sensitive dyes. We assessed δ in human and pig myocardium at excitation/emission wavelengths of commonly used and recently developed near-infrared voltage-sensitive dyes. We also compare δ in Tyrode vs blood-perfused tissues and simulate respective voltage-sensitive fluorescent signals in the context of potential clinical applications (in vivo optical mapping of heart electrical activity). Experiments were conducted in isolated slabs of ventricular myocardium. Light decay inside the tissue was measured via a 600μm diameter optrode at 520, 650, and 715nm. The δ was determined by fitting data to a theoretical formula for light attenuation [Mitrea et al. 2009].View Large Image | View Hi-Res Image | Download PowerPoint SlideFor wavelengths tested, (see table) the δ in porcine and human myocardium are similar, which makes porcine myocardium a good model for development of clinical imaging applications. Blood perfusion reduces δ, particularly at 520nm. However, our simulations show that, the resulting reduction of optical action potential is <16% (for NIR dyes). which would not be a major impediment for in vivo imaging of cardiac excitation.

Comparison of Action Potential Characteristics from Intact Rabbit Myocardium Using 2-Photon Excitation, Widefield Epifluorescence and Microelectrode Recordings

2-photon excitation of voltage-sensitive dyes enables determination of sub-cellular electrical activity within intact myocardium at a range of depths. The investigation aimed to compare action potential (AP) characteristics derived from sub-cellular 2-photon imaging in an intact cardiac preparation using Di-4-ANEPPS with epifluorescence and microelectrode voltage recordings. Hearts from male New Zealand white rabbits were Langendorff-perfused at 37°C and paced at a cycle length of 350ms. Preparations were loaded with Di-4-ANEPPS and optical APs recorded using widefield epifluorescence and 2-photon (2P) microscopy. BDM (10mM) and Blebbistatin (10µM) were used to eliminate motion artefacts. Membrane potentials were recorded at 26KHz before and after perfusion of mechanical uncouplers. Using surface microelectrodes Vmax values of up to 120 V.s−1 were recorded. Mean 10-90% AP rise times were 2.78±0.29 ms (mean±S.E.M) using microelectrodes (n=3), compared with 3.91±0.30 ms recorded at 50 µm below tissue surface with 2P imaging (at 2.6KHz). Matching sampling frequency between microelectrode and 2P recordings abolished this difference. Perfusion of mechanical uncouplers did not significantly alter rise time (2.44±0.20 ms vs. 2.78±0.29 ms; before vs. after, P=0.4) or APD90 (134.90±4.13 ms vs. 130.25±2.93 ms; before vs. after, P=0.4). Mean AP rise times for 2P recordings lengthened with increasing tissue depth (3.91±0.30 ms vs. 5.35±0.23 ms; 50 vs. 250µm from surface, P<0.05, n=7) while epifluorescence rise times were consistently longer (7.85±0.32 ms, n=7). Composite images from multiple depths using 2P microscopy displayed rise times approaching those recorded from widefield epifluorescence. These data suggest that tissue light-scattering at increasing depths results in lengthening of measured AP rise times without significantly altering APD. Slower rise times combined with more diffuse images obtained at depths greater than 200µm suggests optical aberrations cause loss of signal resolution at these depths.

Enhanced Dispersion of Repolarization Explains Increased Arrhythmogenesis in Severe Versus Therapeutic Hypothermia

Hypothermia is proarrhythmic, and, as the use of therapeutic hypothermia (TH) increases, it is critically important to understand the electrophysiological effects of hypothermia on cardiac myocytes and arrhythmia substrates. We tested the hypothesis that hypothermia-enhanced transmural dispersion of repolarization (DOR) is a mechanism of arrhythmogenesis in hypothermia. In addition, we investigated whether the degree of hypothermia, the rate of temperature change, and cooling versus rewarming would alter hypothermia-induced arrhythmia substrates. Optical action potentials were recorded from cells spanning the transmural wall of canine left ventricular wedge preparations at baseline (36°C), during cooling and during rewarming. Electrophysiological parameters were examined while varying the depth of hypothermia. On cooling to 26°C, DOR increased from 26±4 ms to 93±18 ms (P=0.021); conduction velocity decreased from 35±5 cm/s to 22±5 cm/s (P=0.010). On rewarming to 36°C, DOR remained prolonged, whereas conduction velocity returned to baseline. Conduction block and reentry was observed in all severe hypothermia preparations. Ventricular fibrillation/ventricular tachycardia was seen more during rewarming (4/5) versus cooling (2/6). In TH (n=7), cooling to 32°C mildly increased DOR (31±6 to 50±9, P=0.012), with return to baseline on rewarming and was associated with decreased arrhythmia susceptibility. Increased rate of cooling did not further enhance DOR or arrhythmogenesis. Hypothermia amplifies DOR and is a mechanism for arrhythmogenesis. DOR is directly dependent on the depth of cooling and rewarming. This provides insight into the clinical observation of a low incidence of arrhythmias in TH and has implications for protocols for the clinical application of TH.