The long-term goal of this Bioengineering Research Partnership (BRP) is to develop a High-Speed, Depth-Resolved Imager (HSDRI) to map electrical activity or intracellular free Ca 2+ transients inside the myocardium of perfused hearts. The partnership consists of 3 groups. Dr. Guy Salama (PI at the University of Pittsburgh) will administer the BRP, develop the instrument and apply the new technology to problems in cardiac electrophysiology, that remain unresolved due to a lack of 3-D information. Drs. Alan Waggoner (at Carnegie-Mellon University, Director of the Center for Light Microscope Imaging and Biotechnology (CLMIB)) and Lauren Ernst will develop optical probes (voltage-sensitive and Ca2+ indicator dyes) with long excitation and emission wavelengths to improve tissue penetration and reduce light scattering from the myocardium. Dr. Fred Lanni (at CLMIB) will provide the theoretical and engineering expertise to develop and refine the HSDRI. The 3 groups will work in parallel.
Aim 1 (Salama and Lanni, years 1-5): Two approaches will be developed and tested to obtain the best possible HSDRI system. (a) A system based on a Ronchi line grating to focus dark and bright bands in a focal plane 2-5 mm deep in ventricular tissue. Fluorescence images from the tissue will be taken (at 3k frames/s) during shifts of bright and dark bands of light excitation by 1/3 period. Images will be processed on-line to eliminate light emanating above and below the plane of focus to obtain depth-resolved images, at 1k frames/s. (b) A standard Nipkow spinning disk confocal imager will be modified for large fields-of-view (3x3 mm 2)and high frame rates.
Aim 2 (years 1-5): Drs. Waggoner and Ernst will synthesize new longer wavelength fluorescent dyes to monitor action potentials (APs) or cytosolic free Ca2+(Cai) and Dr. Salama will test, analyze the spectral characteristics and response characteristics of the new probes in heart muscle.
Aim 3 (Salama, Choi and Lanni years 1-5): Software will be developed to drive the HSDRI, analyze APs and Ca-i transients and map electrical activity in 3-D. Depth-resolved maps of activation, repolarization and AP durations will be used to investigate 2 topics in cardiac electrophysiology, where measurements in 3-D are essential to elucidate fundamental concepts. A) We will investigate the factors that modify electrical coupling (time-delay or block) between Purkinje fibers (P), Transitional (T) and Ventricular (V) cells to elucidate the role of PV junctions in the initiation and maintenance of arrhythmias. APs will be mapped in 3-D to resolve PV delays during antegrade and retrograde conduction, normoxic and ischemic in paced and during arrhythmias. B) Impulse propagation across the atrio-ventricular node (AVN) has been difficult to trace because of the complex 3-D structure of the node and the small region of compact cells. Activation maps of the AVN in 3-D will help us answer basic questions regarding the precise inputs to the node (fast and slow pathways), mechanisms of AVN reentry, Wenckebach periodicity and Wolf-Parkinson syndrome. Fast, depth-resolved images of voltage and Ca 2+are a powerful new tool that will have a wide range of applications in cardiac electrophysiology and can be extended to neuronal networks and other organ systems. We focus here on the heart because therein lie salient problems that are ready to be addressed by this new technology. However, the wide range of possible applications may lead to the commercialization of this new technology.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-ECS (50))
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Lathrop, David A
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University of Pittsburgh
Schools of Medicine
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N?mec, Jan; Kim, Jong J; Salama, Guy (2016) The link between abnormal calcium handling and electrical instability in acquired long QT syndrome--Does calcium precipitate arrhythmic storms? Prog Biophys Mol Biol 120:210-21
Kim, Jong J; Yang, Lei; Lin, Bo et al. (2015) Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells. J Mol Cell Cardiol 81:81-93
Chen, Guojun; Yang, Xiaoyan; Kerchner, Laurie J et al. (2010) WITHDRAWN: Sex-differences in sodium/calcium exchange expression is a determinant of the arrhythmia phenotype in pre-pubertal rabbit hearts with Long QT type 2. Heart Rhythm :
N?mec, Jan; Kim, Jong J; Gabris, Beth et al. (2010) Calcium oscillations and T-wave lability precede ventricular arrhythmias in acquired long QT type 2. Heart Rhythm 7:1686-94
Ng, G Andre; Mantravadi, RajKumar; Walker, William H et al. (2009) Sympathetic nerve stimulation produces spatial heterogeneities of action potential restitution. Heart Rhythm 6:696-706
Salama, Guy; Hwang, Seong-min (2009) Simultaneous optical mapping of intracellular free calcium and action potentials from Langendorff perfused hearts. Curr Protoc Cytom Chapter 12:Unit 12.17
Sims, Carl; Reisenweber, Steven; Viswanathan, Prakash C et al. (2008) Sex, age, and regional differences in L-type calcium current are important determinants of arrhythmia phenotype in rabbit hearts with drug-induced long QT type 2. Circ Res 102:e86-100
Tallini, Yvonne N; Brekke, Johan Fredrik; Shui, Bo et al. (2007) Propagated endothelial Ca2+ waves and arteriolar dilation in vivo: measurements in Cx40BAC GCaMP2 transgenic mice. Circ Res 101:1300-9
Patrick, Michael J; Ernst, Lauren A; Waggoner, Alan S et al. (2007) Enhanced aqueous solubility of long wavelength voltage-sensitive dyes by covalent attachment of polyethylene glycol. Org Biomol Chem 5:3347-53
Choi, Bum-Rak; Jang, Woncheol; Salama, Guy (2007) Spatially discordant voltage alternans cause wavebreaks in ventricular fibrillation. Heart Rhythm 4:1057-68

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