Bogdan G. Mitrea

Imaging electrical excitation inside the myocardial wall

Cardiac arrhythmias are often triggered by ectopic membrane depolarization originating deep inside the myocardial wall. Here we propose a new method utilizing a novel near-infrared voltage-sensitive fluorescent dye DI-4-ANBDQBS to determine the three-dimensional (3D) coordinates of the sources of such depolarization. We tested the method in live preparations of pig left and right ventricular myocardium (thickness 8-18 mm) and phantoms imitating the optical properties of myocardial tissue. The method utilizes an alternating transillumination approach that involves comparing pairs of simultaneously recorded broad-field epifluorescence and transillumination images produced at two alternating directions of illumination. Recordings were taken simultaneously by two CCD cameras facing the endocardial and epicardial surfaces of the heart at a frame rate up to 3 KHz. In live preparations, we were able to localize the origin of the depolarization wave with a precision of ±1.3mm in the transmural direction and 3 mm in the image plane. The accuracy of detection was independent of the depth of the source inside ventricular wall.

Monitoring intramyocardial reentry using alternating transillumination

Intramyocardial reentry is implicated as a primary cause of the most deadly cardiac arrhythmias known as polymorphic ventricular tachycardia and ventricular fibrillation. However, the mechanisms involved in the triggering of such reentry and controlling its subsequent dynamics remain poorly understood. One of the major obstacles has been a lack of adequate tools that would enable 3D imaging of electrical excitation and reentry inside thick ventricular wall. Here, we present a new experimental technique, termed alternating transillumination (AT), aimed at filling this gap. The AT technique utilizes a recently synthesized near-infrared fluorescent voltage-sensitive dye, DI-4-ANBDQBS. We apply AT to study the dynamics of reentry during shock-induced polymorphic ventricular tachycardia in pig myocardium.

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.

Probing Field-Induced Tissue Polarization Using Transillumination Fluorescent Imaging

Despite major successes of biophysical theories in predicting the effects of electrical shocks within the heart, recent optical mapping studies have revealed two major discrepancies between theory and experiment: 1), the presence of negative bulk polarization recorded during strong shocks; and 2), the unexpectedly small surface polarization under shock electrodes. There is little consensus as to whether these differences result from deficiencies of experimental techniques, artifacts of tissue damage, or deficiencies of existing theories. Here, we take advantage of recently developed near-infrared voltage-sensitive dyes and transillumination optical imaging to perform, for the first time that we know of, noninvasive probing of field effects deep inside the intact ventricular wall. This technique removes some of the limitations encountered in previous experimental studies. We explicitly demonstrate that deep inside intact myocardial tissue preparations, strong electrical shocks do produce considerable negative bulk polarization previously inferred from surface recordings. We also demonstrate that near-threshold diastolic field stimulation produces activation of deep myocardial layers 2-6 mm away from the cathodal surface, contrary to theory. Using bidomain simulations we explore factors that may improve the agreement between theory and experiment. We show that the inclusion of negative asymmetric current can qualitatively explain negative bulk polarization in a discontinuous bidomain model.

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.

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.