Nicoletta C. Surdo

PKA and PDE4D3 anchoring to AKAP9 provides distinct regulation of cAMP signals at the centrosome

Control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals through PKA and PDE4D3 interaction with the A kinase anchoring protein AKAP9.

FRET biosensor uncovers cAMP nano-domains at β-adrenergic targets that dictate precise tuning of cardiac contractility.

Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals. This nanoscopic heterogeneity of cAMP signals is necessary to optimize cardiac contractility upon adrenergic activation. At low adrenergic levels, and those mimicking heart failure, differential local cAMP responses are exacerbated, with near abolition of cAMP signalling at certain locations. This work provides tools and fundamental mechanistic insights into subcellular adrenergic signalling in normal and pathological cardiac function.

Efficacy of B-Type Natriuretic Peptide Is Coupled to Phosphodiesterase 2A in Cardiac Sympathetic Neurons

Elevated B-type natriuretic peptide (BNP) regulates cGMP-phosphodiesterase activity. Its elevation is regarded as an early compensatory response to cardiac failure where it can facilitate sympathovagal balance and cardiorenal homeostasis. However, recent reports suggest a paradoxical proadrenergic action of BNP. Because phosphodiesterase activity is altered in cardiovascular disease, we tested the hypothesis that BNP might lose its efficacy by minimizing the action of cGMP on downstream pathways coupled to neurotransmission. BNP decreased norepinephrine release from atrial preparations in response to field stimulation and also significantly reduced the heart rate responses to sympathetic nerve stimulation in vitro. Using electrophysiological recording and fluorescence imaging, BNP also reduced the depolarization evoked calcium current and intracellular calcium transient in isolated cardiac sympathetic neurons. Pharmacological manipulations suggested that the reduction in the calcium transient was regulated by a cGMP/protein kinase G pathway. Fluorescence resonance energy transfer measurements for cAMP, and an immunoassay for cGMP, showed that BNP increased cGMP, but not cAMP. In addition, overexpression of phosphodiesterase 2A after adenoviral gene transfer markedly decreased BNP stimulation of cGMP and abrogated the BNP responses to the calcium current, intracellular calcium transient, and neurotransmitter release. These effects were reversed on inhibition of phosphodiesterase 2A. Moreover, phosphodiesterase 2A activity was significantly elevated in stellate neurons from the prohypertensive rat compared with the normotensive control. Our data suggest that abnormally high levels of phosphodiesterase 2A may provide a brake against the inhibitory action of BNP on sympathetic transmission.

PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling

cAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death. DOI: http://dx.doi.org/10.7554/eLife.21374.001

The subcellular localization of neuronal nitric oxide synthase determines the downstream effects of NO on myocardial function

Aims In healthy hearts, the neuronal nitric oxide synthase (nNOS) is predominantly localized to the sarcoplasmic reticulum (SR), where it regulates the ryanodine receptor Ca2+ release channel (RyR2) and phospholamban (PLB) phosphorylation, and to a lesser extent to the sarcolemmal membrane where it inhibits the L-type Ca2+ current (ICa). However, in failing hearts, impaired relaxation and depressed inotropy are associated with a larger proportion of nNOS being localized to the sarcolemmal membrane. Whether there is a causal relationship between altered myocardial function and subcellular localization of nNOS remains to be assessed. Methods and results Adenoviruses (AdV) encoding for a human nNOS.eGFP fusion protein or eGFP were injected into the left ventricle (LV) of nNOS−/− mice. nNOS.eGFP localized to the sarcolemmal and t-tubular membrane and immunoprecipitated with syntrophin and caveolin-3 but not with RyR2. Myocardial transduction of nNOS.eGFP resulted in a significantly increased NOS activity (10-fold, P < 0.01), a 20% increase in myocardial tetrahydrobiopterin (BH4) (P < 0.05), and a 30% reduction in superoxide production (P < 0.001). LV myocytes transduced with nNOS.eGFP showed a significantly lower basal and &bgr;-adrenergic stimulated ICa, [Ca2+]i transient amplitude and cell shortening (vs. eGFP). All differences between groups were abolished after NOS inhibition. In contrast, nNOS.eGFP had no effect on RyR nitrosylation, PLB phosphorylation or the rate of myocardial relaxation and [Ca2+]i decay. Conclusion Our findings indicate that nNOS-mediated regulation of myocardial excitation–contraction (E–C) coupling is exquisitely dependent on nNOS subcellular localization and suggests a partially adaptive role for sarcolemmal nNOS in the human failing myocardium.

Adenoviral transduction of FRET-based biosensors for cAMP in primary adult mouse cardiomyocytes.

Genetically encoded biosensors that make use of fluorescence resonance energy transfer (FRET) are important tools for the study of compartmentalized cyclic nucleotide signaling in living cells. With the advent of germ line and tissue-specific transgenic technologies, the adult mouse represents a useful tool for the study of cardiovascular pathophysiology. The use of FRET-based genetically encoded biosensors coupled with this animal model represents a powerful combination for the study of cAMP signaling in live primary cardiomyocytes. In this chapter, we describe the steps required during the isolation, viral transduction, and culture of cardiomyocytes from an adult mouse to obtain reliable expression of genetically encoded FRET biosensors for the study of cAMP signaling in living cells.

Adrenaline Stimulates Glucagon Secretion by Tpc2-Dependent Ca2+ Mobilization From Acidic Stores in Pancreatic α-Cells.

Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of β-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca2+ and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca2+]i in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca2+ but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca2+]i in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca2+ release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca2+]i. Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that β-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca2+]i signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca2+ release from the acidic stores and further amplified by Ca2+-induced Ca2+ release from the sarco/endoplasmic reticulum.

Phosphatases control PKA-dependent functional microdomains at the outer mitochondrial membrane

Significance The selective phosphorylation of spatially distinct PKA targets is key for the pleiotropy of the cAMP cascade. This characteristic of the pathway is currently attributed to the ability of phosphodiesterases or adenylate cyclases to create subcellular sites (microdomains) where the concentration of cAMP is distinct from that of the surrounding areas. The role of phosphatases in this process has not been tested. Here we show that limited access of phosphatases to the PKA targets present at the outer mitochondrial membrane generates distinct microdomains of PKA phosphorylated proteins despite there being no differences in the local cAMP levels. These results describe an alternative mechanism capable of generating functional cAMP/PKA-dependent microdomains and may be extrapolated to the compartmentalization of other kinase-dependent events. Evidence supporting the heterogeneity in cAMP and PKA signaling is rapidly accumulating and has been largely attributed to the localization or activity of adenylate cyclases, phosphodiesterases, and A-kinase–anchoring proteins in different cellular subcompartments. However, little attention has been paid to the possibility that, despite homogeneous cAMP levels, a major heterogeneity in cAMP/PKA signaling could be generated by the spatial distribution of the final terminators of this cascade, i.e., the phosphatases. Using FRET-based sensors to monitor cAMP and PKA-dependent phosphorylation in the cytosol and outer mitochondrial membrane (OMM) of primary rat cardiomyocytes, we demonstrate that comparable cAMP increases in these two compartments evoke higher levels of PKA-dependent phosphorylation in the OMM. This difference is most evident for small, physiological increases of cAMP levels and with both OMM-located probes and endogenous OMM proteins. We demonstrate that this disparity depends on differences in the rates of phosphatase-dependent dephosphorylation of PKA targets in the two compartments. Furthermore, we show that the activity of soluble phosphatases attenuates PKA-driven activation of the cAMP response element-binding protein while concurrently enhancing PKA-dependent mitochondrial elongation. We conclude that phosphatases can sculpt functionally distinct cAMP/PKA domains even in the absence of gradients or microdomains of this messenger. We present a model that accounts for these unexpected results in which the degree of PKA-dependent phosphorylation is dictated by both the subcellular distribution of the phosphatases and the different accessibility of membrane-bound and soluble phosphorylated substrates to the cytosolic enzymes.

Reflections of Research: Flower from the heart, by Dr Nicoletta Surdo

The British Heart Foundation (BHF) annual image competition, Reflections of Research, provides a glimpse into the cutting-edge research that the BHF funds

REGULATION OF cAMP SIGNALS AT INDIVIDUAL MACROMOLECULAR COMPLEXES IN CARDIAC MYOCYTES

The cAMP\PKA pathway regulates a wide range of cellular processes in the heart. cAMP compartmentalization has been demonstrated by FRET-based sensors in cardiac cell, although studies so far have suffered from limited spatial resolution of the imaging approach. The local control of cAMP signals in distinct signalling hubs is of critical importance to achieve specific functions. We developed a new set of tools, based on a novel FRET-tag, named INF2, for quantitative imaging of cAMP capable of dissecting compartmentalized cAMP signalling. Due to its novel design, the INF2 sensor can be targeted to individual macromolecular complexes by fusion to targeting proteins, retaining FRET efficiency independently of the protein it is fused to. These targeted sensors allow accurate quantification and comparison of cAMP fluctuations confined to specific signalling hubs. Expression of the targeted sensors in neonatal and in adult rat cardiomyocytes shows the expected specific targeting. The FRET response to catecholamine stimulation of INF2 fusions to AKAP79, AKAP18d and TnI, three proteins involved in the regulation of excitation-contraction coupling, shows that cAMP signals are uniquely regulated in the microenvironment surrounding these three hubs. Moreover the FRET signal generated by these sensors upon PGE2 and GLP-1 stimulation shows different cAMP responses in different plasma membrane compartments. Such differences are abolished by pretreatment with the PDE inhibitor IBMX, emphasizing the key role of phosphodiesterases in the control of the local cAMP levels. Our results indicate that these new tools have the potential to dissect of cAMP microdomains with unprecedented spatial resolution.