Research May 9 , 2014 Application of GECI in the Heart

Conceptual Considerations Excitation–contraction coupling is an essential mechanism responsible for the blood-pumping function of the heart. In cardiac myocytes, repetitive Ca cycling translates electric excitation (the action potential) into force generation (contraction). Altered Ca handling has been related to many cardiac diseases, such as cardiac hypertrophy and heart failure. Therefore, the elucidation of the initiation and progression of cardiac diseases via monitoring Ca handling, either at the cellular level or in tissue, is of great interest. Fluorescence-based intracellular Ca imaging is the most versatile method for monitoring Ca concentrations in living cells or tissues. Fluorescence-based Ca imaging typically uses Ca-sensitive fluorescent dyes, such as small organic molecules and, to a lesser extent, genetically encoded Ca indicators (GECIs). GECIs represent a new generation of Casensing molecules. The first exogenous expression of green fluorescent protein (GFP) in living cells led to the proposed use of GFPs in genetically encoded biosensors. They offer a multitude of advantages over small molecule indicators, including the following: (1) almost no unintended compartmentalization; (2) homogeneous expression in cells from tissues, enabling imaging at the tissue/organ level; (3) cell-type–specific Ca mapping through promoter-driven expression; (4) subcellular targeting by the expression of fusion proteins; and (5) the possibility for extended imaging over days and weeks, enabling the investigation of chronic treatments. The basic initial concept of the GECI is the coupling of 2 fluorescent proteins of distinct colors to a Ca-binding domain such that the emission spectrum of the so-called donor and the excitation spectrum of the acceptor protein overlap. A conformational change in the Ca-binding domain after Ca binding changes the steric arrangement between the 2 proteins. If the donor is excited, part of its energy can be transferred to the acceptor in a distance-dependent manner by a process called Förster Resonance Energy Transfer (FRET). This idea was realized quickly by several groups, and subsequently, genetically encoded Ca sensors based on such FRET pairs were introduced. A second design approach of GECI is based on circular permutated fluorescent proteins (CPFPs) that lead to the simultaneous development of several of such sensors. Here, the Ca-sensing domain is the same as for FRET sensors. The particular differences in the properties of FRET sensors and CPFP-based sensors will be described in the following sections. Depending on the incorporated fluorescence protein(s) and the Ca-sensing domain, available GECIs exhibit different properties (eg, signal:noise ratio, Ca affinities, or other general properties, such as the Ca detection principle) and require particular read-out modes. Furthermore, a recent report highlighted that some indicators may display cell-type–specific behaviors; therefore, results obtained in one cell system may not necessarily be transferable to other cell types. Here, we provide an overview of the concepts, experiences, state of the art, and ongoing developments in GECI, with particular emphasis on applications in cardiac cells and heart tissue. We also address numerous fundamental concerns, such as obtaining the expression of GECI in cardiac myocytes, comparing Review