Richard D. Walton

Dual excitation wavelength epifluorescence imaging of transmural electrophysiological properties in intact hearts

BACKGROUND Epifluorescence imaging using voltage-sensitive dyes has provided unique insights into cardiac electrical activity and arrhythmias. However, conventional dyes use blue-green excitation light, which has limited depth penetration. OBJECTIVE The aim of this study was to demonstrate that combining a short and a long excitation wavelength using near-infrared (NIR) dyes allows for epifluorescence imaging of transmural electrophysiological properties in intact hearts. METHODS Epifluorescence imaging was performed in rat hearts (N = 11) using DI-4-ANEPPS and the NIR dye DI-4-ANBDQBS. Activation and action potential duration (APD) patterns were investigated at 2 excitation wavelengths (530 and 660 nm) after epicardial stimulation at various cycle lengths (160 to 70 ms). RESULTS Optical action potential upstrokes acquired with 660-nm excitation of DI-4-ANBDQBS were significantly longer than upstrokes obtained with 530-nm excitation of DI-4-ANEPPS (P < .001). Comparison of activation maps showed counterclockwise rotation of isochrones consistent with a transmural rotation of myofibers. Pronounced APD modulation by the activation sequence was observed at both excitation wavelengths. Significantly prolonged APDs (P = .016) and steeper APD restitution curves were found with DI-4-ANBDQBS (660-nm excitation) when compared with DI-4-ANEPPS (530-nm excitation). Dual excitation wavelength experiments using solely DI-4-ANBDQBS yielded similar results. Monophasic action potential recordings showed prolonged APD and steeper APD restitution curves in the endocardium, indicating that 660-nm excitation provides a significant endocardial contribution to the signal. Three-dimensional computer simulations confirmed our findings. CONCLUSION Dual excitation wavelength epifluorescence allows detecting transmural heterogeneity in intact hearts. It therefore has the potential to become an important tool in experimental cardiac electrophysiology.

Construction and validation of anisotropic and orthotropic ventricular geometries for quantitative predictive cardiac electrophysiology

Reaction–diffusion computational models of cardiac electrophysiology require both dynamic excitation models that reconstruct the action potentials of myocytes as well as datasets of cardiac geometry and architecture that provide the electrical diffusion tensor D, which determines how excitation spreads through the tissue. We illustrate an experimental pipeline we have developed in our laboratories for constructing and validating such datasets. The tensor D changes with location in the myocardium, and is determined by tissue architecture. Diffusion tensor magnetic resonance imaging (DT-MRI) provides three eigenvectors ei and eigenvalues λi at each voxel throughout the tissue that can be used to reconstruct this architecture. The primary eigenvector e1 is a histologically validated measure of myocyte orientation (responsible for anisotropic propagation). The secondary and tertiary eigenvectors (e2 and e3) specify the directions of any orthotropic structure if λ2 is significantly greater than λ3—this orthotropy has been identified with sheets or cleavage planes. For simulations, the components of D are scaled in the fibre and cross-fibre directions for anisotropic simulations (or fibre, sheet and sheet normal directions for orthotropic tissues) so that simulated conduction velocities match values from optical imaging or plunge electrode experiments. The simulated pattern of propagation of action potentials in the models is partially validated by optical recordings of spatio-temporal activity on the surfaces of hearts. We also describe several techniques that enhance components of the pipeline, or that allow the pipeline to be applied to different areas of research: Q ball imaging provides evidence for multi-modal orientation distributions within a fraction of voxels, infarcts can be identified by changes in the anisotropic structure—irregularity in myocyte orientation and a decrease in fractional anisotropy, clinical imaging provides human ventricular geometry and can identify ischaemic and infarcted regions, and simulations in human geometries examine the roles of anisotropic and orthotropic architecture in the initiation of arrhythmias.

Quantification of the Transmural Dynamics of Atrial Fibrillation by Simultaneous Endocardial and Epicardial Optical Mapping in an Acute Sheep Model

Background—Therapy strategies for atrial fibrillation based on electric characterization are becoming viable personalized medicine approaches to treat a notoriously difficult disease. In light of these approaches that rely on high-density surface mapping, this study aims to evaluate the presence of 3-dimensional electric substrate variations within the transmural wall during acute episodes of atrial fibrillation. Methods and Results—Optical signals were simultaneously acquired from the epicardial and endocardial tissue during acute fibrillation in ovine isolated left atria. Dominant frequency, regularity index, propagation angles, and phase dynamics were assessed and correlated across imaging planes to gauge the synchrony of the activation patterns compared with paced rhythms. Static frequency parameters were well correlated spatially between the endocardium and the epicardium (dominant frequency, 0.79±0.06 and regularity index, 0.93±0.009). However, dynamic tracking of propagation vectors and phase singularity trajectories revealed discordant activity across the transmural wall. The absolute value of the difference in the number, spatial stability, and temporal stability of phase singularities between the epicardial and the endocardial planes was significantly >0 with a median difference of 1.0, 9.27%, and 19.75%, respectively. The number of wavefronts with respect to time was significantly less correlated and the difference in propagation angle was significantly larger in fibrillation compared with paced rhythms. Conclusions—Atrial fibrillation substrates are dynamic 3-dimensional structures with a range of discordance between the epicardial and the endocardial tissue. The results of this study suggest that transmural propagation may play a role in atrial fibrillation maintenance mechanisms.

Extracting Surface Activation Time from the Optically Recorded Action Potential in Three-Dimensional Myocardium

Optical mapping has become an indispensible tool for studying cardiac electrical activity. However, due to the three-dimensional nature of the optical signal, the optical upstroke is significantly longer than the electrical upstroke. This raises the issue of how to accurately determine the activation time on the epicardial surface. The purpose of this study was to establish a link between the optical upstroke and exact surface activation time using computer simulations, with subsequent validation by a combination of microelectrode recordings and optical mapping experiments. To simulate wave propagation and associated optical signals, we used a hybrid electro-optical model. We found that the time of the surface electrical activation (t(E)) within the accuracy of our simulations coincided with the maximal slope of the optical upstroke (t(F)*) for a broad range of optical attenuation lengths. This was not the case when the activation time was determined at 50% amplitude (t(F50)) of the optical upstroke. The validation experiments were conducted in isolated Langendorff-perfused rat hearts and coronary-perfused pig left ventricles stained with either di-4-ANEPPS or the near-infrared dye di-4-ANBDQBS. We found that t(F)* was a more accurate measure of t(E) than was t(F50) in all experimental settings tested (P = 0.0002). Using t(F)* instead of t(F50) produced the most significant improvement in measurements of the conduction anisotropy and the transmural conduction time in pig ventricles.

Electrophysiological and structural determinants of electrotonic modulation of repolarization by the activation sequence.

Spatial dispersion of repolarization is known to play an important role in arrhythmogenesis. Electrotonic modulation of repolarization by the activation sequence has been observed in some species and tissue preparations, but to varying extents. Our study sought to determine the mechanisms underlying species- and tissue-dependent electrotonic modulation of repolarization in ventricles. Epi-fluorescence optical imaging of whole rat hearts and pig left ventricular wedges were used to assess epicardial spatial activation and repolarization characteristics. Experiments were supported by computer simulations using realistic geometries. Tight coupling between activation times (AT) and action potential duration (APD) were observed in rat experiments but not in pig. Linear correlation analysis found slopes of −1.03 ± 0.59 and −0.26 ± 0.13 for rat and pig, respectively (p < 0.0001). In rat, maximal dispersion of APD was 11.0 ± 3.1 ms but dispersion of repolarization time (RT) was relatively homogeneous (8.2 ± 2.7, p < 0.0001). However, in pig no such difference was observed between the dispersion of APD and RT (17.8 ± 6.1 vs. 17.7 ± 6.5, respectively). Localized elevations of APD (12.9 ± 8.3%) were identified at ventricular insertion sites of rat hearts both in experiments and simulations. Tissue geometry and action potential (AP) morphology contributed significantly to determining influence of electrotonic modulation. Simulations of a rat AP in a pig geometry decreased the slope of AT and APD relationships by 70.6% whereas slopes were increased by 75.0% when implementing a pig AP in a rat geometry. A modified pig AP, shortened to match the rat APD, showed little coupling between AT and APD with greatly reduced slope compared to the rat AP. Electrotonic modulation of repolarization by the activation sequence is especially pronounced in small hearts with murine-like APs. Tissue architecture and AP morphology play an important role in electrotonic modulation of repolarization.

Influence of the Purkinje-muscle junction on transmural repolarization heterogeneity

AIMS To elucidate the properties of the PMJ and myocardium underlying these effects. Transmural heterogeneity of action potential duration (APD) is known to play an important role in arrhythmogenesis. Regions of non-uniformities of APD gradients often overlap considerably with the location of Purkinje-muscle junctions (PMJs). We therefore hypothesized that such junctions are novel sources of local endocardial and transmural heterogeneity of repolarization, and that remodelling due to heart failure modulates this response. METHODS AND RESULTS Spatial gradients of endocardial APD in left ventricular wedge preparations from healthy sheep (n = 5) were correlated with locations of PMJs identified through Purkinje stimulation under optical mapping. APD prolongation was dependent on proximity of the PMJ to the imaged surface, whereby shallow PMJs significantly modulated local APD when stimulating either Purkinje (P = 0.0116) or endocardium (P = 0.0123). In addition, we model a PMJ in 5 × 5× 10 mm transmural tissue wedges using healthy and novel failing human ventricular and Purkinje ionic models. Short distances of the PMJ to cut surfaces (<0.875 mm) revealed that APD maxima were localized to the PMJ in healthy myocardium, whereas APD minima were observed in failing myocardium. Amplitudes and spatial gradients of APD were prominent at functional PMJs and quiescent PMJs. Furthermore, increasing the extent of Purkinje fibre branching or decreasing tissue conductivity augmented local APD prolongation in both failing and non-failing models. CONCLUSIONS The Purkinje network has the potential to influence myocardial AP morphology and rate-dependent behaviour, and furthermore to underlie enhanced transmural APD heterogeneities and spatial gradients of APD in non-failing and failing myocardium.

Transmural electrophysiological heterogeneity, the T-wave and ventricular arrhythmias.

Heterogeneous distribution of electrophysiological behavior across the heart is essential for normal cardiac function. If this heterogeneity becomes excessive it may contribute to arrhythmogenesis and sudden cardiac death. Controversy exists regarding the localization of activation and repolarization gradients in the diseased heart and how these heterogeneities contribute to arrhythmogenesis. In this review we focus on the genesis and existence of transmural heterogeneity in activation and repolarization. We will describe a possible embryonic origin of these heterogeneities and address the question how heterogeneities contribute to the genesis of the electrocardiogram and how they may cause reentrant arrhythmias. This review subsequently concentrates on several pathologies in which transmural heterogeneities are thought to play a role.

A novel near-infrared voltage-sensitive dye reveals the action potential wavefront orientation at increased depths of cardiac tissue

Recently, novel near-infrared (NIR) voltage-sensitive dyes were developed for imaging electrical activity in blood-perfused hearts and for tomographic applications. However, their usefulness for conventional surface mapping is unclear. The spectral shift to the NIR range significantly increases the penetration depth of light into the tissue, thus increasing the intramural volume contributing to the optical action potential (OAP). Here, we characterize both computationally and experimentally the effect of increased penetration depth on the OAP upstroke, the OAP component most sensitive to optical scattering and absorption, and the activation maps. Optical imaging of cardiac electrical activity was performed in isolated rat hearts (n = 3D5) paced from the LV mid free wall. We used the NIR dye JPW-6033 (excitation at 660nm, acquisition at >695nm). The conventional dye DI-4-ANEPPS (excitation at 532nm, acquisition at 700 DF50nm) was used for comparison. To simulate OAP we utilized a hybrid model that couples light transport equations with the model of electrical propagation. As expected, the switch from DI-4-ANEPPS to JPW-6033 significantly increased the upstroke duration: from 3.95±0.69ms to 5.39±0.82 ms, respectively. However, activation maps were largely unaffected. The correlation between the shape of the optical upstroke, and the averaged subsurface wave front orientation was also preserved. The computer simulations are in excellent agreement with the experimental data. In conclusion, our analysis suggests that despite significant increase in upstroke duration, the novel NIR dyes can be a useful alternative to conventional dyes in surface mapping applications.

A technical review of optical mapping of intracellular calcium within myocardial tissue

Optical mapping of Ca(2+)-sensitive fluorescence probes has become an extremely useful approach and adopted by many cardiovascular research laboratories to study a spectrum of myocardial physiology and disease conditions. Optical mapping data are often displayed as detailed pseudocolor images, providing unique insight for interpreting mechanisms of ectopic activity, action potential and Ca(2+) transient alternans, tachycardia, and fibrillation. Ca(2+)-sensitive fluorescent probes and optical mapping systems continue to evolve in the ongoing effort to improve therapies that ease the growing worldwide burden of cardiovascular disease. In this technical review we provide an updated overview of conventional approaches for optical mapping of Cai (2+) within intact myocardium. In doing so, a brief history of Cai (2+) probes is provided, and nonratiometric and ratiometric Ca(2+) probes are discussed, including probes for imaging sarcoplasmic reticulum Ca(2+) and probes compatible with potentiometric dyes for dual optical mapping. Typical measurements derived from optical Cai (2+) signals are explained, and the analytics used to compute them are presented. Last, recent studies using Cai (2+) optical mapping to study arrhythmias, heart failure, and metabolic perturbations are summarized.

Interactions of Short-Term and Chronic Treadmill Training With Aging of the Left Ventricle of the Heart

With aging, there is a decline in cardiac function accompanying increasing risk of arrhythmias. These effects are likely to be mechanistically associated with age-associated changes in calcium regulation within cardiac myocytes. Previous studies suggest that lifelong exercise can potentially reduce age-associated changes in the heart. Although exercise itself is associated with changes in cardiac function, little is known about the interactions of aging and exercise with respect to myocyte calcium regulation. To investigate this, adult (12 months) and old (24 months) C57/Bl6 mice were trained using moderate-intensity treadmill running. In response to 10 weeks’ training, comparable cardiac hypertrophic responses were observed, although aging independently associated with additional cardiac hypertrophy. Old animals also showed increased L- and T-type calcium channels, the sodium–calcium exchange, sarcoendoplasmic reticulum calcium ATPase, and collagen (by 50%, 92%, 66%, 88%, and 113% respectively). Short-term exercise training increased D-type and T-type calcium channels in old animals only, whereas an increase in sodium–calcium exchange was seen only in adult animals. Long-term (12 months) training generally opposed the effects of aging. Significant hypertrophy remained in long-term trained old animals, but levels of sarcoendoplasmic reticulum calcium ATPase, sodium–calcium exchange, and collagen were not significantly different from those found in the adult trained animals.