Abstract
Studies in isolated cardiomyocytes have provided tremendous information at the cellular and molecular level concerning regulation of transmembrane voltage (Vm) and intracellular calcium ([Ca2+]i). The ability to use the information gleaned to gain insight into the function of ion channels and Ca2+ handling proteins in a more complex system, e.g., the intact heart, has remained a challenge. We have developed laser scanning fluorescence microscopy-based approaches to monitor, at the sub-cellular to multi-cellular level in the immobilized, Langendorff-perfused mouse heart, dynamic changes in [Ca2+]i and Vm. This article will review the use of single- or dual-photon laser scanning microscopy [Ca2+]i imaging in conjunction with transgenic reporter technology to (a) interrogate the extent to which transplanted, donor-derived myocytes or cardiac stem cell-derived de novo myocytes are capable of forming a functional syncytium with the pre-existing myocardium, using entrainment of [Ca2+]i transients by the electrical activity of the recipient heart as a surrogate for electrical coupling, and (b) characterize the Ca2+ handling phenotypes of cellular implants. Further, we will review the ability of laser scanning fluorescence microscopy in conjunction with a fast-response voltage-sensitive to resolve, on a subcellular level in Langendorff-perfused mouse hearts, Vm dynamics that typically occur during the course of a cardiac action potential. Specifically, the utility of this technique to measure microscopic-scale voltage gradients in the normal and diseased heart is discussed.
Highlights
Studies in single isolated cardiomyocytes have provided important information at the cellular and molecular level concerning the electrical properties and Ca2+ regulation
This review focuses on the ability of single- or dual-photon laser scanning fluorescence microscopy (i) to assess the function of intracardiac cell transplants and stem cell-derived de novo myocardium, when used in combination with transgenic reporter technology, and (ii) to measure spatiotemporal dispersion of electrical and Ca2+ signals at the microscopic scale in normal and diseased heart
ASSESSMENT OF SPATIOTEMPORAL DISPERSION OF ELECTRICAL AND Ca2+ SIGNALS IN LANGENDORFF-PERFUSED HEARTS USING LASER SCANNING MICROSCOPY To be able to directly assess electrical activity on a microscopic scale in situ, we previously developed an optical assay using laser confocal scanning imaging in conjunction with the fastresponse, voltage-sensitive dye Annine-6plus (Bu et al, 2009)
Summary
Studies in single isolated cardiomyocytes have provided important information at the cellular and molecular level concerning the electrical properties and Ca2+ regulation. Multicellular preparations may be valuable for electrophysiological characterizations, those related to action potential properties and propagation Such preparations would enable the study of key issues about myocyte Ca2+ regulation, including the conditions required for Ca2+ wave initiation and propagation from cell-to-cell. Cardiac physiology/pathophysiology mandates the development of assays capable of resolving dynamic events with cellular/subcellular resolution in intact cardiac tissue. In their pioneering study, Wier and co-workers developed a confocal laser scanning microscopy-based technique to monitor local sarcoplasmic reticulum (SR) Ca2+ release phenomena and the propagation of Ca2+ waves in isolated rat papillary muscles iontophoretically loaded with the calcium indicator fluo-3 (Wier et al, 1997). We previously developed a technique to optically monitor, on a sub- to multi-cellular scale, intracellular calcium ([Ca2+]i) dynamics in the intact, Langendorff-perfused mouse heart, using two-photon laser scanning microscopy (TPLSM) in conjunction with calcium-sensitive fluorescent dyes
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