FRETting mice shed light on cardiac adrenergic signaling.

-adrenergic receptor ( -AR) signaling in cardiac myocytes influences contractile and relaxation states in the heart. Classically, following hormone activation, -AR preferentially couples with Gs, which in turn activates adenylyl cyclase and cAMP production. The predominant effector of cAMP, cAMP-dependent protein kinase (PKA), then phosphorylates many proteins important for cardiac function such as L-type calcium channels and phospholamban in the sarcoplasmic reticulum membrane. However, studies in myocytes have shown not all receptors that transduce signals via cAMP generate the same functional effects.1 These observations have led to the concept of cAMP compartmentation, which attributes the functional specificity and differential regulation to intricate spatial and temporal control of signaling molecules in the cAMP pathway. In this issue of Circulation Research, Nikolaev et al2 take another look at the differentially regulated 1 and 2-adrenergic signaling by using FRET-based cAMP imaging in adult cardiomyocytes, a method that is well suited for revealing the spatiotemporal complexity in cAMP signaling. Fluorescence ratio imaging of cAMP in living cells was first introduced 15 years ago with the development of a bimolecular indicator using fluorescent dye-tagged regulatory and catalytic subunits of PKA.3 This class of cAMP indicators has evolved to become genetically encodable with the use of green fluorescent protein (GFP) variants4,5 and in recent years, unimolecular with the use of single cAMP binding domain-containing proteins and protein fragments.6–8 Nikolaev et al add to our molecular toolbox with the development of yet another FRET-based cAMP sensor with new characteristics. This sensor uses the cAMP binding domain of the hyperpolarization-activated cyclic nucleotide gated channel 2 (HCN2) as opposed to the binding domains from PKA or exchange proteins directly activated by cAMP (Epac), used in other FRET sensors. The resulting biosensor, HCN2-camps, maintains a high sensitivity for cAMP but does not appear to saturate at physiological cAMP concentrations in adult cardiomyocytes, thus being able to report agonistinduced changes in cAMP. Another important characteristic of the HCN2-camps is its uniform distribution in cardiomyocytes that allows for the measurement of cAMP dynamics throughout the entire cell without bias from sensor localization. A significant point illustrated by design and application of this new cAMP biosensor is the importance of being able to use a tailored cAMP biosensor that is most suited for a particular experiment, cell system, or functional study. Several key characteristics are worth considering when designing or choosing a cAMP sensor. First, the binding property of the sensor, such as binding affinity for cAMP, is among the most important characteristics. Different cell types have varying basal levels of cAMP and different capacity of cAMP production and degradation, thus requiring use of biosensors that have appropriate detection ranges that match concentrations of endogenous cAMP. Secondly, expression levels and localization patterns vary between different sensors. For example, a PKA-based cAMP sensor showed distinct subcellular localization within cardiomyocytes because of interaction with A kinase anchoring proteins (AKAPs).5 Furthermore, biosensors can be targeted to various subcellular sites in the cell for examining specific pools of cAMP and signaling microdomains,7 whereas a diffusible indicator is more suited for visualizing global cAMP changes. Thirdly, the dynamic range of a sensor refers to the difference in fluorescence signals, usually reported as emission ratios for this class of sensors, between cAMP-bound and unbound states. A large dynamic range is always a desirable property for achieving high signal to noise ratios, particularly in the case of detecting subtle and suboptimal changes. Other parameters, such as fluorescent properties of the FRET pair, could also be tailor-designed to accommodate specific application needs. Fortunately, an array of cAMP biosensors is already available that expresses a combination of characteristics, which are often complementary to one another. It can be expected that new variants with improved or novel properties will be further developed to provide revealing windows into the complex cAMP regulation in various live systems. The live systems suitable for fluorescence imaging range from cells to tissues and animals. Although a popular choice and good model systems in many cases, cultured cell lines are usually far removed from their tissue origin and may exhibit an altered signaling environment such as altered surface expression of receptors and remodeling of innate cellular architecture. Primary cells, which may be more physiologically relevant, are often difficult to transfect. The introduction of viral vectors for gene transduction has pushed research past this obstacle. However, the expression of fluorescent protein-based probes in primary cells usually requires longer culturing to insure a highly expressed sensor with matured The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Departments of Pharmacology and Molecular Sciences (J.Z., L.M.D.), and Neuroscience (J.Z.), The Johns Hopkins University School of Medicine, Baltimore, Md. Correspondence to Jin Zhang, PhD, Departments of Pharmacology and Molecular Sciences, and Neuroscience, The Johns Hopkins University School of Medicine, Rm 307 Hunterian Bldg, 725 N. Wolfe Street, Baltimore, MD 21205. E-mail [email protected] (Circ Res. 2006;99:1021–1023.) © 2006 American Heart Association, Inc.