Cyclic nucleotide imaging and cardiovascular disease

Abstract

The universal second messengers cyclic nucleotides 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) play central roles in cardiovascular function and disease. They act in discrete, functionally relevant subcellular microdomains which regulate, for example, calcium cycling and excitation-contraction coupling. Such localized cAMP and cGMP signals have been difficult to measure using conventional biochemical techniques. Recent years have witnessed the advent of live cell imaging techniques which allow visualization of these functionally relevant second messengers with unprecedented spatial and temporal resolution at cellular, subcellular and tissue levels. In this review, we discuss these new imaging techniques and give examples how they are used to visualize cAMP and cGMP in physiological and pathological settings to better understand cardiovascular function and disease. Two primary techniques include the use of Förster resonance energy transfer (FRET) based cyclic nucleotide biosensors and nanoscale scanning ion conductance microscopy (SICM). These methods can provide deep mechanistic insights into compartmentalized cAMP and cGMP signaling.

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.