Cyclic nucleotide imaging and cardiovascular disease
Introduction
Cyclic nucleotides 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are ubiquitous second messengers which integrate upstream neurohormonal signals sensed by membrane or intracellular receptors to regulate a myriad of physiological effector functions, including cell metabolism, development, memory formation, hormone secretion, cardiac contractility, platelet activation and vascular tone (Beavo & Brunton, 2002). In the heart, cAMP generated in response to catecholamine stimulation of cardiomyocyte (CM) β-adrenergic receptors (β-ARs) modulates excitation-contraction coupling by cAMP-dependent protein kinase (PKA)-mediated phosphorylation of several calcium handling and contractile proteins. This helps the heart to meet an increased contractility demand upon physical or emotional stress. However, chronic stimulation of the cAMP signaling pathway leads to maladaptive cardiac remodelling (Lohse, Engelhardt, & Eschenhagen, 2003). cGMP is generally considered as a “protective” second messenger since pharmacological elevation of cGMP levels by either inhibition of cGMP hydrolyzing phosphodiesterases (PDEs) or activation of cGMP synthesis by soluble and particulate guanylyl cyclases protects the heart from hypertrophy and ischemic injury (Boerrigter et al., 2009, Korkmaz et al., 2009, Lee et al., 2015, Takimoto et al., 2005). While PKA and cGMP-dependent protein kinase (cGK) often phosphorylate similar substrates, their functional effects in CMs can be vastly different. To explain this puzzling fact and the specificity of cyclic nucleotide responses, a theory of cyclic nucleotide compartmentation has emerged that is now commonly accepted. This paradigm and the approaches to visualize compartmentalized cAMP and cGMP signals in functionally relevant CM microdomains are the main focus of this review. We will discuss new imaging technologies and give examples how they have advanced our understanding of cardiac function and disease.
Section snippets
Cyclic nucleotides and their role in the heart
Stimulation of G-protein coupled receptors (GPCRs) such as β-ARs at the CM membrane act via G-proteins and adenylyl cyclases (ACs), enzymes which convert adenosine triphosphate to cAMP, to increased or decreased cAMP synthesis. The main CM intracellular effectors of cAMP are PKA and exchange protein directly activated by cAMP (Epac) that are ubiquitously expressed and cyclic nucleotide-gated (CNG) channels that are present almost exclusively in sinoatrial node myocytes. CNG channels are
Cyclic nucleotide microdomains
Very early studies using classical biochemical methods and later investigations using electrophysiological and live cell imaging approaches have established intracellular compartmentation as an important mechanism conferring specificity and efficiency in cyclic nucleotide signaling. Early studies involving cell fractionation demonstrated that activation of cells with various receptor ligands such as the β-AR agonist isoproterenol (ISO) and prostaglandin E1 results in elevations of cAMP in
cAMP imaging using FRET-based biosensors
To directly visualize cAMP and cGMP action in their specific subcellular microdomains, extensive scientific and methodological development has been required. It was not until the advent of modern live cell imaging techniques that a more clear picture of local cyclic nucleotide signaling and its alteration in cardiac disease started to emerge (Berrera et al., 2008, Lefkimmiatis and Zaccolo, 2014, Sprenger and Nikolaev, 2013).
Real time cAMP dynamics in living cells can be visualized using several
Live cell cGMP imaging
Reliable measurement of cGMP in cells and tissues, especially in CMs, has been challenging (Götz & Nikolaev, 2013). In the last decade, several optical and non-optical methods to measure cGMP in single intact cells have been developed. One important example is electrophysiological recording using ectopically expressed cyclic nucleotide-gated (CNG) channels as sensors for subsarcolemmal cGMP. This method uncovered the differential contributions of PDE families in cGMP compartmentation in adult
Imaging cyclic nucleotides microdomains in health and disease
To target cAMP biosensors to different functionally relevant microdomains, several Epac1-camps fusion sensors have been developed. One elegant study fused this sensor to dimerization-docking domains of regulatory type I (RI) and type II (RII) PKA subunits for targeting to intracellular sites where endogenous PKA type I and II are located. These sensors showed that β-AR stimulation led to a selective increase of cAMP in RII-associated microdomains, whereas prostaglandin receptors stimulated
Nanoscale imaging by FRET in combination with scanning ion conductance microscopy
To gain even more precise insights into microdomain-specific cyclic nucleotide signaling and to study receptor-microdomains interactions, a combination of FRET biosensors with SICM can be used as a state-of-the-art nanoscale imaging technique (Miragoli et al., 2011, Nikolaev et al., 2010).
Initially, this combination was developed to answer an important question regarding where β1- and β2-ARs are localized on the surface of adult cardiomyocytes with respect to the underlying, highly organized
cAMP imaging in intact hearts
The majority of scientific data on cyclic nucleotide compartmentation and microdomain specific signaling have been obtained in vitro using isolated and electrically non-stimulated CMs. Recently, using paced myocytes, we demonstrated that FRET measurements in quiescent CMs under β-adrenergic stimulation and PDE4 inhibition were representative of cAMP responses obtained in electrically-stimulated contracting myocytes (Sprenger, Bork, Herting, Fischer, & Nikolaev, 2016). However, it remains
Conflict of interest statement
The authors declare no conflicts of interest.
Funding
The work in the authors' laboratory is supported by the grants from the German Research Foundation (“Deutsche Forschungsgemeinschaft” grants NI 1301/1, NI 1301/3, and FOR 2060) and by the Gertraud und Heinz-Rose Stiftung.
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