Valentina Lissandron

Fluorescence Resonance Energy Transfer–Based Analysis of cAMP Dynamics in Live Neonatal Rat Cardiac Myocytes Reveals Distinct Functions of Compartmentalized Phosphodiesterases

Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes. By performing real-time imaging of cAMP in situ, we establish the hierarchy of these PDEs in controlling cAMP levels in basal conditions and on stimulation with a β-adrenergic receptor agonist. PDE4, rather than PDE3, appears to be responsible for modulating the amplitude and duration of the cAMP response to beta-agonists. PDE3 and PDE4 localize to distinct compartments and this may underpin their different functional roles. Our findings indicate the importance of distinctly localized PDE isoenzymes in determining compartmentalized cAMP signaling.

Compartmentalized Phosphodiesterase-2 Activity Blunts β-Adrenergic Cardiac Inotropy via an NO/cGMP-Dependent Pathway

&bgr;-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP]i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving &bgr;3-adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by &bgr;-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the &bgr;-adrenergic pathway. In this, activation of &bgr;3-adrenergic receptors counteracts cAMP generation obtained via stimulation of &bgr;1/&bgr;2-adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.

Protein kinase A type I and type II define distinct intracellular signaling compartments.

Protein kinase A (PKA) is a key regulatory enzyme that, on activation by cAMP, modulates a wide variety of cellular functions. PKA isoforms type I and type II possess different structural features and biochemical characteristics, resulting in nonredundant function. However, how different PKA isoforms expressed in the same cell manage to perform distinct functions on activation by the same soluble intracellular messenger, cAMP, remains to be established. Here, we provide a mechanism for the different function of PKA isoforms subsets in cardiac myocytes and demonstrate that PKA-RI and PKA-RII, by binding to AKAPs (A kinase anchoring proteins), are tethered to different subcellular locales, thus defining distinct intracellular signaling compartments. Within such compartments, PKA-RI and PKA-RII respond to distinct, spatially restricted cAMP signals generated in response to specific G protein–coupled receptor agonists and regulated by unique subsets of the cAMP degrading phosphodiesterases. The selective activation of individual PKA isoforms thus leads to phosphorylation of unique subsets of downstream targets.

AKAP complex regulates Ca2+ re‐uptake into heart sarcoplasmic reticulum

The β‐adrenergic receptor/cyclic AMP/protein kinase A (PKA) signalling pathway regulates heart rate and contractility. Here, we identified a supramolecular complex consisting of the sarcoplasmic reticulum Ca2+‐ATPase (SERCA2), its negative regulator phospholamban (PLN), the A‐kinase anchoring protein AKAP18δ and PKA. We show that AKAP18δ acts as a scaffold that coordinates PKA phosphorylation of PLN and the adrenergic effect on Ca2+ re‐uptake. Inhibition of the compartmentalization of this cAMP signalling complex by specific molecular disruptors interferes with the phosphorylation of PLN. This prevents the subsequent release of PLN from SERCA2, thereby affecting the Ca2+ re‐uptake into the sarcoplasmic reticulum induced by adrenergic stimuli.

Restricted diffusion of a freely diffusible second messenger: mechanisms underlying compartmentalized cAMP signalling.

It is becoming increasingly evident that the freely diffusible second messenger cAMP can transduce specific responses by localized signalling. The machinery that underpins compartmentalized cAMP signalling is only now becoming appreciated. Adenylate cyclases, the enzymes that synthesize cAMP, are localized at discrete parts of the plasma membrane, and phosphodiesterases, the enzymes that degrade cAMP, can be targeted to selected subcellular compartments. A-kinase-anchoring proteins then serve to anchor PKA (protein kinase A) close to specific targets, resulting in selective activation. The specific activation of such individual subsets of PKA requires that cAMP is made available in discrete compartments. In this presentation, the molecular and structural mechanisms responsible for compartmentalized PKA signalling and restricted diffusion of cAMP will be discussed.

Unique characteristics of Ca2+ homeostasis of the trans-Golgi compartment

Taking advantage of a fluorescent Ca2+ indicator selectively targeted to the trans-Golgi lumen, we here demonstrate that its Ca2+ homeostatic mechanisms are distinct from those of the other Golgi subcompartments: (i) Ca2+ uptake depends exclusively on the activity of the secretory pathway Ca2+ ATPase1 (SPCA1), whereas the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) is excluded; (ii) IP3 generated by receptor stimulation causes Ca2+ uptake rather than release; (iii) Ca2+ release can be triggered by activation of ryanodine receptors in cells endowed with robust expression of the latter channels (e.g., in neonatal cardiac myocyte). Finally, we show that, knocking down the SPCA1, and thus altering the trans-Golgi Ca2+ content, specific functions associated with this subcompartment, such as sorting of proteins to the plasma membrane through the secretory pathway, and the structure of the entire Golgi apparatus are dramatically altered.

Ca2+ signalling in the Golgi apparatus

The Golgi apparatus plays a central role in lipid and protein post-translational modification and sorting. Morphologically the organelle is heterogeneous and it is possible to distinguish stacks of flat cysternae (cis- and medial Golgi), tubular-reticular networks and vesicles (trans-Golgi). These morphological differences parallel a distinct functionality with a selective distribution and complementary roles of the enzymes found in the different compartments. The Golgi apparatus has been also shown to be involved in Ca(2+) signalling: it is indeed endowed with Ca(2+) pumps, Ca(2+) release channels and Ca(2+) binding proteins and is thought to participate in determining the spatio-temporal complexity of the Ca(2+) signal within the cell, though this role is still poorly understood. Recently, it has been demonstrated that the organelle is heterogeneous in terms of Ca(2+) handling and selective reduction of Ca(2+) concentration, both in vitro and in a genetic human disease, within one of its sub-compartment results in alterations of protein trafficking within the secretory pathway and of the entire Golgi morphology. In this paper we review the available information on the Ca(2+) toolkit within the Golgi, its heterogeneous distribution in the organelle sub-compartments and discuss the implications of these characteristics for the physiopathology of the Golgi apparatus.

Compartmentalized cAMP/PKA signalling regulates cardiac excitation–contraction coupling

The sympathetic control over excitation–contraction coupling (ECC) is mediated by the cAMP/PKA signalling pathway. However, in the myocyte, the same signalling pathway is responsible for triggering a plethora of diverse intracellular functions the control of which must be independent of the regulation of ECC. Here we discuss what are the molecular mechanisms leading to selective modulation of ECC in cardiac myocytes with a particular focus on the role of spatial confinement of PKA subsets and the compartmentalization of cAMP.

Imaging the cAMP-dependent signal transduction pathway.

In recent years, the development of new technologies based on the green fluorescent protein and FRET (fluorescence resonance energy transfer) has introduced a new perspective in the study of cAMP signalling. Real-time imaging of fluorescent biosensors is making it possible to visualize cAMP dynamics directly as they happen in intact, living cells, providing important and original insights for our understanding of the spatiotemporal organization of the cAMP/PKA (protein kinase A) signalling pathway.

Heterogeneity of Ca2+ handling among and within Golgi compartments

The Golgi apparatus (GA) is a dynamic intracellular Ca(2+) store endowed with complex Ca(2+) homeostatic mechanisms in part distinct from those of the endoplasmic reticulum (ER). We describe the generation of a novel fluorescent Ca(2+) probe selectively targeted to the medial-Golgi. We demonstrate that in the medial-Golgi: (i) Ca(2+) accumulation takes advantage of two distinct pumps, the sarco/endoplasmic reticulum Ca(2+) ATPase and the secretory pathway Ca(2+) ATPase1; (ii) activation of IP3 or ryanodine receptors causes Ca(2+) release, while no functional two-pore channel was found; (iii) luminal Ca(2+) concentration appears higher than that of the trans-Golgi, but lower than that of the ER, suggesting the existence of a cis- to trans-Golgi Ca(2+) concentration gradient. Thus, the GA represents a Ca(2+) store of high complexity where, despite the continuous flow of membranes and luminal contents, each sub-compartment maintains its Ca(2+) identity with specific Ca(2+) homeostatic characteristics. The functional role of such micro-heterogeneity in GA Ca(2+) handling is discussed.