University of Groningen Understanding compartmentalized cAMP signaling for potential therapeutic approaches in cardiac disease Musheshe, Nshunge

(1)

Understanding compartmentalized cAMP signaling for potential therapeutic approaches in

cardiac disease

Musheshe, Nshunge

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.

Document Version

Publisher's PDF, also known as Version of record

Publication date: 2018

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):

Musheshe, N. (2018). Understanding compartmentalized cAMP signaling for potential therapeutic

approaches in cardiac disease: Insights into the molecular mechanisms of the cAMP-mediated regulation of the cardiac phospholemman-Na+/K+ ATPase complex. University of Groningen.

Copyright

Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

(2)

520589-L-bw-Nshunge 520589-L-bw-Nshunge 520589-L-bw-Nshunge 520589-L-bw-Nshunge Processed on: 26-6-2018 Processed on: 26-6-2018 Processed on: 26-6-2018

Processed on: 26-6-2018 PDF page: 7PDF page: 7PDF page: 7PDF page: 7 1 Chapt er 1

Chapter 1 General Introduction

(3)

Processed on: 26-6-2018 PDF page: 8PDF page: 8PDF page: 8PDF page: 8 2

Chapt

er

1

PREFACE

The primary objective of this work was to study how cAMP-PKA signaling coordinates ion flux at the plasmalemma of the cardiac myocytes by using Fluorescence Resonance Energy Transfer (FRET) based sensors. In this general introduction, I focus on the importance of cAMP-PKA signaling and its

compartmentalization in cardiac myocytes in the regulation of Ca2+_{in the heart.}

After presenting the principle functions of regulating Ca2+ _{homeostasis, role of}

Na+_{channels and their regulators in health and disease, subsequently tools to}

demonstrate cAMP-PKA compartmentalization are described and the interplay

between the L-type Ca2+_{channels/A kinase Anchoring Protein 79(AKAP79) and}

Phospholemman-Na+_/K+_{ATPase in cardiac myocytes is emphasized.}

CYCLIC AMP (cAMP): FUNCTIONS

3’,5’-cyclic adenosine monophosphate (cAMP) is produced by the catalytic conversion of adenosine triphosphate (ATP), by a plasma membrane bound enzyme - adenylyl cyclase (AC). ACs are lyases that convert ATP to cAMP and pyrophosphate. cAMP is a small and diffusible universal second messenger which converts the signal carried by extracellular stimuli into specific cellular responses. For example, cAMP transfers into cells the effects of hormones such as glucagon and adrenaline which cannot pass through the cell membrane thereby enabling regulation of glycogen, sugar and lipid metabolism. Because of the role of cAMP in various cellular responses such as metabolism, electrical activity and transcription regulation, the signal transduction pathway of cAMP has been intensely studied since the cyclic nucleotide’s discovery in 1953 by Earl Sutherland (Berthet J et al., 1957).

(4)

Chapt

er

1

cAMP exerts its functions via activation of its effectors: protein kinase A (PKA) (Taylor S.S et al., 1992), the exchange protein directly activated by cAMP (Epac) (Schmidt M et al., 2013; Desaubry L et al., 1996), cyclic nucleotide-gated (CNG) ion channels (Kaupp U.B., Seifert R., 2002; Biel, M and Michalakis S., 2009) and most recently a novel class of three-pass transmembrane popeye domain containing proteins (Popdc proteins) which bind cAMP with a high affinity has also been described (Roland F.R et al., 2016). By activating these intracellular targets, cAMP controls cellular functions ranging from frequency and strength of cardiac contraction to cell growth and differentiation to cell movement and migration (Francis, S.H and Corbin, J.D et al., 1994).

COMPARTMENTALIZATION OF cAMP-PKA

SIGNALING

The ability of cAMP to regulate a large variety of cellular functions in a specific manner results from the organization of an intricate and complex network of signaling pathways which coexist within each single cell and operate under strict spatial and temporal control. It has been found that individual pools of cAMP affect a limited subset of PKA targets thereby mediating different functional effects and acquiring specificity of signaling (Stangherlin, A and Zaccolo, M., 2012). Gs protein coupled receptors (GPCRs), ACs, and cAMP effectors are spatially organized in macromolecular complexes, or signalosomes, to allow selective phosphorylation of targets in response to specific stimuli (Buxton I.L.O and Brunton L.L., 1983; Zaccolo M and Pozzan T., 2002).

In the heart, for example, cAMP-dependent PKA mediates the catecholaminergic control over the force and frequency of cardiac contraction via phosphorylation of proteins that are involved in excitation-contraction coupling (ECC). As part of

(5)

Chapt

er

1

the ECC machinery, catecholamine dependent activation of PKA on β-adrenergic

stimulation leads to phosphorylation of the L-type Ca2+_{channels (LTCC) and}

phospholamban (PLB) resulting in increased systolic Ca2+_{and positive inotropy,}

while PKA-mediated phosphorylation of Troponin I (TPNI) reduces the affinity

of the myofilament for Ca2+_{, seemingly nullifying the effect of increased [Ca}2+_]

i.

Thus, PKA activation appears to mediate opposing effects on intracellular Ca2+

levels and how the myocyte coordinates these apparently divergent effects of

adrenergic stimulation on [Ca2+_]

i remains unclear.

Research over the past fifteen years indicates that compartmentalization is a key feature of cAMP signaling. Compartmentalization of signaling components allows for the signal from individual extracellular stimuli to propagate inside the cell along defined and specific pathways within the network (Stangherlin A, Zaccolo M., 2012). For example, it was demonstrated that Isoproterenol (ISO) generates a cAMP pool that only activates PKA type II while prostaglandin1 (PGE1) generates a cAMP pool that only activates PKA type I. This discovery revealed that independent compartments exist within the cell where cAMP levels are uniquely regulated (Di Benedetto, G et al., 2008).

COMPARTMENTALIZATION OF cAMP BY

PHOSPHODIESTERASES (PDE)S

The cAMP signal is terminated by the action of metallohydrolases called phosphodiesterases (PDEs), which are enzymes that hydrolyze cAMP into inactive 5’-AMP (Conti, M and Beavo, J., 2007). PDEs are subdivided into 11 families (namely: PDE1-11) that are encoded by 21 different genes. Some of these PDE families (PDE-1,-2,-3,-4,-5,-7,-8 and -9) have been identified in the cardiovascular system. PDEs 1,2,3,4,7 and 8 degrade cAMP while PDE5 and 9

(6)

Chapt

er

1

degrade only cyclic GMP. PDE2 and PDE3 are dual-specific enzymes and can degrade both cAMP and cGMP. The expression and distribution of PDEs vary among species (Francis, S.H et al., 2011). Over 100 PDE enzyme variants are formed from multiple promoters due to alternative splicing. Partly due to the existence of multiple splice variants which vary in their expression profile, drug targeting of a specific subset of PDEs is currently a theme in pharmaceutical drug discovery programs (Maurice D.H et al., 2014).

By degrading cAMP within specific subcellular domains and thus acting as sinks for cAMP, PDEs contribute to its dishomogenous distribution within the cell. As a consequence, the cAMP response to an extracellular stimulus is compartmentalized in stimulus-specific sub-microscopic domains (Surdo N.C et

al., 2017;Kokkonen K., Kass D.A., 2017; Zaccolo, M and Pozzan., 2002). PDEs

are distributed inside the cell at particular and critical sites which enables them to regulate local cAMP dynamics in space and in time (Houslay, M.D., 2010; Stangherlin,A and Zaccolo, M., 2011) and regulate the localization, duration and amplitude of cAMP signaling within subcellular domains. This spatial and temporal regulation of cAMP by PDEs allows for activation of certain subsets of PKA in proximity of target proteins required to be phosphorylated, and it prevents unnecessary phosphorylation of proteins at other sites (Terrin, A et al., 2006).

Depending on the specific needs of the cell, presence and activity of individual PDEs at different sites may change. PDEs can be released and/or recruited from specific signaling complexes at different times and at different locations (Housley M.D., 2010). Different tissue distribution and intracellular localization of PDE

isoforms is determined in part by the NH2-terminal domains. The localized

(7)

Chapt

er

1

of cAMP signals have been shown to be involved in the pathogenesis of a number of conditions including hypertrophy, arrhythmia and heart failure (HF), among others (El-Armouche A, and Eschenhagen T., 2009). Plethora of research as a consequence therefore, focusing on developing drugs that target PDEs and PDE inhibition has been used in the treatment of erectile dysfunction, chronic obstructive pulmonary disease (COPD) and acute heart failure among others (Maurice D.H et al., 2014).

COMPARTMENTALIZATION OF PKA BY A KINASE

ANCHORING PROTEINS (AKAPS)

cAMP-PKA signaling pathway is also compartmentalized by A kinase-anchoring proteins (AKAPs), a group of structurally diverse proteins, which are capable of binding to PKA and organizing the PKA into macromolecular complexes (Lohmann, S.M et al., 1986., Sarkar, D et al., 1984). This in turn allows for activation of specific subsets of PKA and the subsequent phosphorylation of select protein targets in response to cAMP-raising stimuli (Steinberg S.F and Brunton L.L., 2001; Dodge-kafka K.L et al., 2006).

Since their discovery, at least 20 different genes encoding for AKAPs have been identified and cloned. There are about 50 members in the AKAP family. All members of the AKAP family share a conserved PKA anchoring amphipathic α-helix of 14-18 residues (Newlon, M.G et al.,1999). PKA and AKAPs interact via an α-helix on the AKAP and a hydrophobic groove formed by the

dimerization/docking domains on the NH2-terminus of PKA R subunits (Gold,

M.G et al., 2006; Kinderman, F.S et al., 2006). Some AKAPs bind preferentially to PKA-RI subunits (RI-selective AKAPs) while others to PKA-RII subunits (RII-selective AKAPs), and others bind to both (dual-specific AKAPs) (Poppinga

(8)

Chapt

er

1

W.J et al., 2014). Functional relevance of PKA anchoring to AKAPs in the heart was demonstrated in previous studies by over expressing Ht31 peptide (known to block binding of PKA to all AKAPs). This resulted in reduced cardiomyocyte shortening due to reduced PKA-dependent phosphorylation of troponin I and myosin binding protein C (Fink, M.A et al., 2001). Also, various AKAPs which play a role in regulation of ECC have been described. One of the most extensively characterized AKAPs is AKAP79/150. AKAP79/150 localizes to the plasmalemma and forms a complex with the LTCC, the β-adrenergic receptor, adenyl cyclase 5/6 and the phosphatases PP1 (Le A.V et al., 2011) and PP2B (Dell’Acqua M.L et al., 2002; Efendiev R et al., 2010). AKAP79 tethers PKA in proximity to the LTCC (Reuter H., 1984) and facilitates its PKA mediated phosphorylation in response to β-adrenergic receptor stimulation, leading to

increased Ca2+_{current and enhanced systolic Ca}2+_{(Gao T et al., 1997). AKAP79/}

LTCCs complexes therefore play a crucial role in generation of normal cardiac rhythm and in triggering atrial and ventricular contraction.

AKAPs also bind to other components beside PKA and these include: PDEs, phosphatases (which dephosphorylate targets downstream of PKA), and also other kinases (Protein Kinase C (PKC) and Mitogen-activated protein kinase (MAPK)). This characteristic allows for the integration and processing of various signals within discreet sites in the cell (Beene, D.L &Scott, J.D., 2007).

THE CARDIAC SODIUM PUMP: NA

+

_/K

+

_ATPASE

The Na+_/K+_{ATPase (NKA) enzyme belongs to the P-type ATPases family of}

proteins which form a characteristic phosphorylated intermediate during the

catalytic cycle (Horisberger, J.D., 2004). The NKA also known as Na+_/K+_pump

(9)

Chapt

er

1

contribute to specialized tissue functions such as regulation of cell volume (Hall JE and Guyton AC., 2006), maintenance of the resting membrane potential and ionic gradients and availing of the electromotive energy for driving numerous transmembrane transport processes that are crucial for normal cellular function (Fuller W and Shattock MJ.,2006). The NKA is located at the plasma membrane

and moves sodium (3 Na+_{) out of the cell, while pumping potassium (2 K}+_{) into}

the cells by using the energy released by hydrolysis of ATP. [Na+_]

i and Na+

transport are key factors for regulation of Ca2+_{cycling, contractility, action}

potential waveform and metabolism in cardiac myocytes (Bers D.M et al.,2003).

In the heart and skeletal muscle cells, the NKA is tightly coupled to the Na+_/Ca2+

exchanger (NCX) which, in its forward mode of action, transports three Na+_ions

inside the cell in exchange for one Ca2+_{ion outside the cell and is the main route}

for Ca2+_{extrusion from cardiac myocytes (Bers D.M 2001). Stimulated NKA sets}

the gradient for the NCX resulting in extrusion of Ca2+_{in exchange of Na}+_{. [Na}+_]

i

is the result of a delicate balance between Na+_{influx and efflux. Perturbation of}

this balance in HF results in elevated [Na+_]

i, with important consequences on

cardiac myocyte function.

[Na+]i is elevated in HF, both in humans and in animal models (Despa S et

al.,2002;Pieske B et al., 2002). By favoring more Ca2+_{influx via NCX, elevated}

[Na+_]

i may limit the contractile dysfunction in HF. However, high [Na+]i may

also negatively affect the cardiac metabolism (McCormack J.G et al., 1990) and the oxidative state of the cell (Kohlhaas M et al., 2010; Despa S and Bers D.M.,

2013). Elevated [Na+_]

i for example accelerates mitochondrial Ca2+ efflux via the

mitochondrial Na+_/Ca2+_{Exchanger and reduces [Ca}2+_]

m (Maack C et al., 2006;

(10)

Chapt

er

1

REGULATION OF THE NA

+

_/K

+

_ATPASE:

Various mechanisms are involved in the regulation of the NKA as a way of enabling proper maintenance of basic cellular and specialized tissue functions. Dysregulation of the pump through defective production or function of tissue-specific regulators has been implicated in various disorders including, but not limited to, cardiovascular diseases, renal and neurological disorders (Laski, M.E and Kurtzman, N.A., 1996; Rose, A.M and Valdes, R., 1994).

A number of mechanisms are involved in the regulation of the NKA to enable the pump to adapt its activity and/or expression to changing physiological demands and allow for proper specialized tissue functions. The regulatory mechanisms include; 1) steroid hormones, for example aldosterone, which affect α and β gene transcription, thereby increasing the number of NKA (Feraille, E and Doucet, A., 2001) and 2) peptide hormones and neurotransmitters which provoke phosphorylation of the cardiac NKA by protein kinases that in turn modulates the pump’s cell surface expression (Therien, A.G and Blostein, R., 2000).

REGULATION OF THE CARDIAC NA

+

_/K

+

_{ATPASE BY}

FXYD1 (PHOSPHOLEMMAN)- THE KINASE TARGET

Small-membrane proteins of the FXYD family were identified as regulators of the NKA. Interaction of FXYD proteins with the NKA modifies the transport properties of the NKA in a tissue-and-isoform specific way instead of changing its expression. FXYD1 also known as phospholemman (PLM) has been shown

(11)

Chapt

er

1

through coimmunoprecipitation experiments as the FXYD protein that interacts/co-localizes with and regulates the cardiac NKA. PLM is a small 72 amino acids sarcolemmal protein which is considered as the major substrate for PKA in the heart (Palmer, C.J et al., 1991). PLM is phosphorylated on specific serine residues by PKA, PKC and the protein kinase never in mitosis A (NIMA) (Lu, K.P et al., 1994; Walaas, S.I et al., 1994). Phosphorylation of serine 68 of PLM by PKA has been implicated in the increased NKA currents measured in forskolin-treated ventricular myocytes (Silverman, B.Z et al., 2005). Protein phosphatase 1 (PP1) has been shown to dephosphorylate PLM at the PKA site S68 and decrease NKA activity (El-Armouche et al., 2011).

Evidence shows that PLM regulates the activity of the cardiac NKA in a PKA-dependent manner and forms an integral part of the NKA complex. PLM provides the link between cAMP-dependent kinase activation and pump modulation (Silvermana, B.Z et al., 2005). Phosphorylation or ablation of PLM relieves

inhibition of the NKA by increasing its Vmax and apparent Na+ affinity (Despa S

et al., 2005; Silverman B.Z et al., 2005; Crambert G et al., 2002; Bibert S et al., 2008). PLM phosphorylation and the subsequent NKA disinhibition are an

integral part of the sympathetic fight-or-flight response, by enhancing Na+

extrusion to better keep up with the increased Na+_{influx at higher heart rates and}

with larger Ca2+_{transients (which drive greater inward Na}+_/Ca2+_Exchanger

(NCX) current) (Despa S et al., 2005), limits the rise in [Na+_]

i during β-adrenergic

stimulation. Thus, NKA regulation by PLM may prevent Ca2+_{overload and}

triggered arrhythmias during sympathetic stimulation of the heart.

On β-adrenergic stimulation, PKA-mediated phosphorylation of PLM enhances

(12)

Chapt

er

1

in turn sets the gradient for NCX and favors extrusion of Ca2+ _{in exchange of Na}+

via the NCX (Fig. 1: panel A), resulting in reduction of [Ca2+_]

i and hence inotropy

(Bers D.M and Despa S., 2009; Despa S. et al.,2008). At apparently the same time, PKA-mediated phosphorylation of LTCC/AKAP79 complex leads to

increased [Ca2+_]

i and positive inotropy (Fig. 1: panel B in green). The mechanisms

that allow coordinated regulation of these apparently opposing effects of PKA

activation on systolic Ca2+_{remain largely to be determined, however by using}

novel fluorescence resonance energy transfer (FRET)-based reporters that are targeted to unique multiprotein complexes it is now possible to demonstrate that cAMP/PKA signaling at various subcellular domains is compartmentalized.

(13)

Chapt

er

1

Figure 1. PKA-mediated phosphorylation of proteins involved in ECC. Panel A: Schematic

representation of NCX gradient set by NKA activity. Panel B: schematic illustration of PKA mediated phosphorylation of proteins involved in cardiac ECC on β-AR stimulation.

(14)

Chapt

er

1

UNRAVELLING cAMP NANODOMAINS USING FRET

TOOLS:

Detection of cAMP in living cells was explored in order to determine how stimulation of different receptors that act via the same cAMP can ensure appropriate cellular responses. It is now known that cAMP is not uniformly distributed through the entire cytoplasm, but it accumulates to a greater or lesser extent in distinct loci within the cell (Schleicher K and Zaccolo M., 2018; Surdo N.C et al., 2017; Zaccolo, M and Pozzan., 2002). The Fluorescence Resonance Energy Transfer (FRET)-based biosensor method which was developed using cyclic nucleotide binding domains allows for real-time visualization of cAMP in an intact cell with high spatial temporal resolution (Zaccolo M and Pozzan T., 2002; Nikolaev, V.O et al., 2004; Surdo N.C et al., 2017). FRET-based sensors have allowed for the demonstration of cAMP compartmentalization (Musheshe N et al., 2018), and as a result have sharpened our ability to detect and monitor cAMP signaling events. Targeting of the sensor to various subcellular domains such as the nuclei or mitochondria allowed for the measurement of the dynamic local changes in cAMP concentrations in response to PGE1 or adrenergic stimulation (DiPilato L.M et al., 2004). Using FRET sensors, it has also been demonstrated that cAMP increase in the cell is heterogenous and in some cases this heterogeneity is due to PDEs which confine cAMP by creating boundaries and prevent its diffusion to other domains (Zaccolo, M and Pozzan, T., 2000; Zaccolo M and Pozzan T et al., 2002; Surdo N.C et al., 2017). By using a cAMP FRET-based sensor, a study by Surdo N.C et al demonstrated that the cardiac response to catecholamines at AKAP18δ/ SERCA/PLB complex at the Sarcoplasmic Reticulum (SR), the AKAP79/β-AR/adenylyl cyclase/LTCC complex at the plasmalemma and the troponin complex at the myoﬁlaments all of which are involved in regulating ECC, was heterogenous. The cAMP signal generated in response to β-AR stimulation differs at these three sites in both

(15)

Chapt

er

1

amplitude and kinetics. On PDE inhibition, equal responses at the three sites were detected, indicating that the heterogeneity depends on local cAMP degradation by PDEs.

cAMP FRET-BASED SENSORS: EPAC1-CAMPS AND

CUTie

In order to address the limitations of the Epac1-camps sensor (Nikolaev V.O et al., 2014), the cAMP Universal Tag for imaging experiments (CUTie) sensor was generated (Surdo N.C et al 2017). The Epac1-camps sensor is comprised of a single amino-and carboxyl termini of the cyclic nucleotide binding domain (CNBD) sandwiched between the FRET pair yellow fluorescent protein and cyan fluorescent protein (YFP-CFP). Targeting domains (TD) are protein components of various multiprotein complexes which when fused to the sensor, direct the sensor to the subcellular site where the targeting domain is normally localized and allow for the detection of cAMP at various subcellular domains in the intact cell. Fusion of Epac1-camps sensor to targeting domains however, affected the sensor’s FRET kinetics with targeted sensors exhibiting variable maximal FRET change due to steric hinderance of the FRET pair by the targeting domain (Surdo N.C et al., 2017).

(16)

Chapt

er

1

Figure 2: A) Schematic representation of targeted Epac1-camps FRET-based sensor. B) Schematic

representation of the CUTie FRET-based sensor. C) Ribbon representation of targeted CUTie in its cAMP-bound (i.e. when FRET occurs) or cAMP-free form (when no FRET occurs). TD is targeting Domain.

In that regard, the CUTie sensor was generated as a way to minimize steric hinderance effects from the targeting domains. The CUTie sensor was designed in a such way that the CFP was fused at the C-terminus of the cAMP binding domain while YFP was inserted into an external and flexible loop of the cAMP binding domain, leaving the N-terminus of the polypeptide chain free for fusion to the targeting domain, that is thus removed from the FRET module and less likely to sterically interfere with the GFP pair (Surdo N.C et al., 2017).

(17)

Chapt

er

1

When tested in intact cells, CUTie shows FRET changes in response to increasing cAMP concentrations. The sensor shows a NO-FRET to FRET response i.e. on binding of cAMP to the cAMP binding domain in CUTie, there is an increase in acceptor emission and a decrease in donor emission due to a decrease in the distance between YFP and CFP. The CUTie sensor shows a FRET change of 15% in the presence of saturating cAMP.

The great advantage of this sensor is the fact that fusion of selected targeting proteins to CUTie does not significantly affect the maximum FRET change and kinetics of the FRET change as the targeting domain and FRET module are physically separated (Surdo N.C et la., 2017). In addition, expression of targeted CUTie reporters in adult and neonatal rat cardiac myocytes shows the expected subcellular localization and conserved responses meaning that the performance of the sensor is not affected by the structural and complex organization of the cell types. Also, the contractile performance of the cell is not affected by overexpression of the sensor. These particular advantages render the CUTie targeted sensors as reliable and suitable tools for the study of cAMP dynamics at individual submicroscopic domains.

PKA FRET-BASED SENSOR: AKAR4

A Kinase activity reporter 4 (AKAR4) sensor – belongs to a family of single-chain FRET sensors which allow for the assay of PKA-mediated phosphorylation downstream of cAMP increase. In the AKAR4 sensor, the phosphorylatable PKA consensus and phosphor-serine binding domain are flanked by a FRET pair of

(18)

Chapt

er

1

fluorophores (Zhang J et al., 2001). When phosphorylated by PKA, the PKA consensus interacts with the phospho-serine-binding domain resulting in conformational change necessary for FRET to occur. The AKAR4 sensor is unique in a sense that it has a high dynamic range and it allows for reversible phosphorylation of the probe (Zhang J et al., 2005; Allen M. D and Zhang J., 2006). The AKAR4 sensor therefore can be used to measure both PKA activity which results in FRET increase and phosphatase activity that dephosphorylates PKA targets and in turn decrease FRET.

Similar to cAMP FRET-based sensors, AKAR4 sensor can be targeted which allows for real-time monitoring of PKA-mediated phosphorylation in a given nanodomain and indirectly provide information on cAMP signaling on its effectors in each nanodomain. Of note however, is that the concentration dependency of AKAR phosphorylation is quite steep (Hill coefficient 2.0) (Koschinski A et al., 2017) and the sensor may saturate at concentrations of cAMP at which the endogenous target of PKA are not fully phosphorylated.

In general, FRET-based tools provide a platform for unraveling cAMP nanodomains and allow for accurate quantification of PKA activity and enable further understanding of the cAMP-regulated nanodomains in cardiomyocytes.

SCOPE OF THE THESIS

Understanding the role of compartmentalization of both cAMP and PKA that ensures distinct responses within the cell has opened up a plethora of possibilities to develop targeted therapies for the treatment of cardiac disease. cAMP/PKA

(19)

Chapt

er

1

signaling ensures the regulation of cytosolic and SR Ca2+ _{which is necessary for}

maintaining cardiac output. Any changes in Ca2+ _{handling may result in}

contraction abnormalities. Advancement in Imaging techniques using highly sensitive FRET sensors that can be targeted to endogenous macromolecular complexes has unraveled the existence of nanodomains in intact cells under heterogenous cAMP pools on β-adrenergic receptor stimulation using various hormones.

Chapter 2

, reviews the evidence in support of cAMP nanodomains and how FRET-based tools have made it possible to study cAMP and PKA activity in real time with high spatial and temporal resolution.

Chapter 3

, explores compartmentalization of cAMP-PKA signaling at

PLM/NKA and LTCC/AKAP79 by using FRET-based sensors and investigates the role of PDEs and phosphatases at the respective nanodomains.

Chapter 4

, investigates whether the use of AKAP5 as a targeting domain for cAMP and PKA FRET-based sensors affects cAMP and PKA activity readouts at the nanodomain. This research explores some shortcomings of targeted sensors and what to keep in mind when expressing proteins that form part of macromolecular complexes like AKAP5 which bind PKA itself.

Chapter 5

, discusses major findings of this work and future directions of each research chapter. The chapter provides perspective on the role this work may play in advancing our understanding of cAMP/PKA compartmentalization in both normal physiology and pathophysiology.

(20)

Processed on: 26-6-2018 PDF page: 25PDF page: 25PDF page: 25PDF page: 25 19 Chapt er 1

REFERENCES:

Allen MD, Zhang J. 2006. Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem Biophys Res Commun 348:716-21

Arystarkhova E, Donnet C, Asinovski NK, Sweadner KJ. 2002. Differential Regulation of Renal Na+_{/ K}+_{ATPase by Splice Variants of the gamma Subunit. J Biol Chem 277:10162–10172}

Attali B, Latter H, Rachamim N, and Garty H. 1995. A corticosteroidinduced gene expressing an “IsK-like” K+_{channel activity in Xenopus oocytes. Proc Natl Acad Sci USA 92: 6092–6096.}

Be´guin P, Crambert G, Monnet-Tschudi F, Uldry M, Horisberger JD, Garty H, and Geering K. 2002. FXYD7 is a brain-specific regulator of Na+_/K+_{ATPase _1-_ isozymes. EMBO J 21: 3264–}

3273.

Beene DL, Scott JD. 2007. A-kinase anchoring proteins take shape. Curr Opin Cell Biol 19:192-8 Berry RG, Despa S, Fuller W, Bers DM, Shattock MJ. 2007. Differential distribution and regulation of mouse cardiac Na+_{/ K}+_{-ATPase alpha1 and alpha2 subunits in T-tubule and surface sarcolemmal}

membranes. Cardiovasc Res 73:92–100

Bers DM, Barry WH, Despa S. 2003. Intracellular Na regulation in cardiac myocytes. Cardiovasc Res.57:897–912.

Bers, DM. 2001. Excitation-contraction coupling and cardiac contractile force. 2. Kluwer Academic Publishers. p. 427

Berthet J et al. 1957. The relationship of epinephrine and glucagon to liver phosphorylase. IV. Effect of epinephrine and glucagon on the reactivation of phosphorylase in liver homogenates. J.Biol.Chem. 224:463-475

Bibert S, Roy S, Schaer D, Horisberger J-D, Geering K. Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes. J Biol Chem. 2008; 283:476–486

Biel M, Michalakis S. Cyclic nucleotide-gated channels. Hand Exp Pharmacol 191: 111–136, 2009. Bossuyt J, Ai X, Moorman JR, Pogwizd SM, Bers DM. 2005. Expression and phosphorylation of the NA-pump regulatory subunit phospholemman in heart failure. Circ Res 97:558–65.

Broillet MC. 2000. A single intracellular cysteine residue is responsible for the activation of the olfactory cyclic nucleotide-gated channel by NO. J Biol Chem 275:15135-41

Buxton ILO, Brunton LL.1983. Compartments of Cyclic-Amp and Protein-Kinase in Mammalian Cardiomyocytes. Journal of Biological Chemistry. 258:233–239.

Conti M, Beavo J. 2007. Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem 76:481-511

(21)

Chapt

er

1

Crambert G, Fuzesi M, Garty H, Karlish S, Geering K. 2002. Phospholemman (FXYD1) associates with Na+,K+-ATPase and regulates its transport properties. Proc Natl Acad Sci USA. 99:11476– 11481.

Dell'Acqua M.L, Dodge K.L, Tavalin S.J, Scott J.D. 2002. Mapping the protein phosphatase-2B anchoring site on AKAP79. Binding and inhibition of phosphatase activity are mediated by residues 315–360. J Biol Chem. 277:48796–48802

Desaubry, L., Shoshani, I., and Johnson, R. A. 1996. J. Biol. Chem. 271: 2380–2382.

Despa S, Bers DM. 2013. Na+_{transport in the normal and failing heart—remember the balance. J}

Mol Cell Cardiol.61:2-10

Despa S, Bossuyt J, Han F, Ginsburg KS, Jia LG, Kutchai H, Tucker AL, Bers DM. 2005. Phospholemman-phosphorylation mediates the β-adrenergic effects on Na/K pump function in cardiac myocytes. Circ Res. 97:252–259.

Despa S, Islam MA, Weber CR, Pogwizd SM, Bers DM. 2002. Intracellular Na+ concentration is elevated in heart failure, but Na/K-pump function is unchanged. Circulation. 105:2543– 2548. DiPilato LM, Cheng X, Zhang J. 2004. Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc Natl Acad Sci U S A 101:16513-8

Dodge-Kafka KL, Langeberg L, Scott JD. 2006. Compartmentation of cyclic nucleotide signaling in the heart - The role of A-kinase anchoring proteins. Circulation Research. 98:993–1001. Efendiev, R. et al. 2010. AKAP79 interacts with multiple adenylyl cyclase (AC) isoforms and scaffolds AC5 and -6 to alpha-amino-3-hydroxyl-5-methyl-4isoxazole-propionate (AMPA) receptors. J. Biol. Chem. 285, 14450–14458.

El-Armouche A, Eschenhagen T. 2009. Beta-adrenergic stimulation and myocardial function in the failing heart. Heart Fail Rev. 14:225–241.

El-Armouche, A., Wittko¨pper, K., Fuller, W., Howie, J., Shattock, M. J., Pavlovic, D. 2011. Phospholemman-dependent regulation of the cardiac Na+/ K+ ATPase activity is modulated by inhibitor-1 sensitive type-1 phosphatase. FASEB J. 25, 4467–4475

Feraille E and Doucet A. 2001. Sodium-potassium-adenosine triphosphatasedependent sodium transport in the kidney: hormonal control. Physiol Rev 81: 345–418.

Fink MA, Zakhary DR, Mackey JA, Desnoyer RW, Apperson- Hansen C, Damron DS, Bond M. 2001. AKAP-mediated targeting of protein kinase A regulates contractility in cardiac myocytes. Circ Res 88: 291– 297.

Forbush B 3rd, Kaplan JH, Hoffman JF. 1978. Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na, K-ATPase. Biochemistry 17:3667–3676

Francis SH, Blount MA, Corbin JD. 2011. Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions. Physiol Rev 91: 651–690.

(22)

Chapt

er

1

Francis SH, Corbin JD. 1994. Structure and function of cyclic nucleotide-dependent protein kinases. Annu Rev Physiol 56: 237– 272

Fuller W, Shattock MJ. 2006. Phospholemman and the Cardiac Sodium Pump. Circ Res.1290-1292. Gao T, Yatani A, Dell’Acqua ML, Sako H, Green SA, Dascal N, Scott, JD, Hosey MM. 1997. cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 19: 185–196.

Geering K. 1991. The functional role of the beta-subunit in the maturation and intracellular transport of Na, K-ATPase. FEBS Lett 285:189–193

Gold MG, Lygren B, Dokurno P, Hoshi N, McConnachie G, Tasken K, Carlson CR, Scott JD, Barford D. 2006. Molecular basis of AKAP specificity for PKA regulatory subunits. Mol Cell 24: 383–395

Hall JE, Guyton AC. 2006. Textbook of medical physiology. St. Louis, Mo: Elsevier Saunders. ISBN 0-7216-0240-1.

Herget, S., Lohse, M. J. & Nikolaev, V. O. 2008. Real-time monitoring of phosphodiesterase inhibition in intact cells. Cell. Signal. 20, 1423–1431.

Horisberger JD. 2004. Recent insights into the structure and mechanism of the sodium pump. Physiology Bethesda.19: 377–387.

Houslay MD. 2010. Underpinning compartmentalised cAMP signaling through targeted cAMP breakdown. Trends Biochem Sci 35: 91–100.

James PF, Grupp IL, Grupp G, Woo AL, Askew GR, Croyle ML, Walsh RA, Lingrel JB. 1999. Identification of a specific role for the Na, K-ATPase a2 isoform as a regulator of calcium in the heart. Mol Cell 3:555–563

Kaupp U.B., Seifert R. 2002. Cyclic nucleotide-gated ion channels. Physiol. Rev. 82:769–824. Kinderman FS, Kim C, von Daake S, Ma Y, Pham BQ, Spraggon G, Xuong NH, Jennings PA, Taylor SS. 2006. A dynamic mechanism for AKAP binding to RII isoforms of cAMP-dependent protein kinase. Mol Cell 24:

Kohlhaas M, Liu T, Knopp A, Zeller T, Ong MF, Böhm M, O’Rourke B, Maack C. 2010. Elevated cytosolic Na+ increases mitochondrial formation of reactive oxygen species in failing cardiac myocytes. Circulation. 121:1606–1613

Kokkonen K., Kass D.A. 2017. Nanodomain Regulation of Cardiac Cyclic Nucleotide Signaling by Phosphodiesterases. Annu. Rev. Pharmacol. Toxicol. 57:455–479.

Koschinski A, Zaccolo M. 2017. Activation of PKA in cell requires higher concentration of cAMP than in vitro: implications for compartmentalization of cAMP signaling. Sci Rep. 7:14090. Krupinski, J., Coussen, F., Bakalyar, H., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R., and Gilman, A. G. 1989. Science 244: 1558–1564

Laski ME and Kurtzman NA. 1996. The renal adenosine triphosphatases— functional integration and clinical significance. Miner Electrolyte Metab 22: 410–422.

(23)

Chapt

er

1

Le, A. V., Tavalin, S. J., & Dodge-Kafka, K. L. 2011. Identification of AKAP79 as a protein phosphatase 1 catalytic binding protein. Biochemistry, 50(23), 5279-5291.

Liu M, Chen TY, Ahamed B, Li J, Yau KW. 1994. Calcium-calmodulin modulation of the olfactory cyclic nucleotide-gated cation channel. Science 266:1348-54

Lohmann SM, Walter U, DeCamilli P. 1986. Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with tissue-specific binding proteins including microtubule-associated proteins. Ann NY Acad Sci 466: 449–452.

Lu KP, Kemp BE, and Means AR. 1994. Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase. J Biol Chem 269: 6603–6607,

Maack C, Cortassa S, Aon MA, Ganesan AN, Liu T, O’Rourke B. 2006. Elevated cytosolic Na+ decreases mitochondrial Ca2+ uptake during excitation-contraction coupling and impairs energetic adaptation in cardiac myocytes. Circ Res.99:172–182

Maurice, D. H. et al. 2014. Advances in targeting cyclic nucleotide phosphodiesterases. Nat. Rev. Drug Discov. 13, 290–314.

McCormack JG, Halestrap AP, Denton RM. 1990. Role of calcium ions in regulation of mammalian intramitochondrial metabolism. Physiol Rev.70:391–425

McDonough AA, Zhang Y, Shin V, Frank JS. 1996. Subcellular distribution of sodium pump isoform subunits in mammalian cardiac myocytes. Am J Physiol 270:C1221–C1227

Morrison BW, Moorman JR, Kowdley GC, Kobayashi YM, Jones LR, and Leder P. 1995. Mat-8, a novel phospholemman-like protein expressed in human breast tumors, induces a chloride conductance in Xenopus oocytes. J Biol Chem 270: 2176–2182,

Newlon MG, Roy M, Morikis D, Hausken ZE, Coghlan V, Scott JD, Jennings PA. 1999. The molecular basis for protein kinase A anchoring revealed by solution NMR. Nat Struct Biol 6: 222– 227.

Nikolaev VO, Bunemann M, Hein L, Hannawacker A, Lohse MJ. 2004. Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215-8

Palmer CJ, Scott BT, and Jones LR. 1991. Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. J Biol Chem 266: 11126–11130,

Pieske B, Maier LS, Piacentino V 3rd, Weisser J, Hasenfuss G, Houser S. Rate dependence of [Na+]i and contractility in nonfailing and failing human myocardium. Circulation. 2002; 106:447– 453

Poppinga WJ, Muñoz-Llancao P, González-Billault C, Schmidt M. 2014. Cyclic AMP compartmentalization in neurodegenerative and obstructive pulmonary diseases, Br J Pharmacol. 24:5603-23.

Rich TC, Fagan KA, Nakata H, Schaak J, Cooper DM, Karpen JW. 2000. Cyclic nucleotide-gated channels colocalize with adenylyl cyclase regions of restricted cAMP diffusion. J Gen Physiol 116:147-61

(24)

Chapt

er

1

Rich TC, Fagan KA, Nakata H, Schaak J, Cooper DM, Karpen JW. 2001a. A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell. Proc Natl Acad Sci USA 98:13049-54

Rich TC, Tse TE, Rohan JG, Schaack J, Karpen JW. 2001b. In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. J Gen Physiol 118:63-78

Roland F. R. Schindler, Chiara Scotton,Vanessa French, Alessandra Ferlini, and Thomas Brand. 2016.The Popeye Domain Containing Genes and Their Function in Striated Muscle. J Cardiovasc Dev Dis. 3(2): 22

Rose AM and Valdes R. 1994. Understanding the sodium pump and its relevance to disease. Clin Chem 40: 1674–1685

Schleicher K and Zaccolo M. 2018. Using cAMP sensors to study cardiac nanodomains. J Cardiovasc Dev Dis. 5(1):17

Schmidt M., Dekker F.J., Maarsingh H. 2013. Exchange Protein Directly Activated by cAMP (epac): A Multidomain cAMP Mediator in the Regulation of Diverse Biological Functions. Pharmacol. Rev. 65:670–709.

Semb SO, Lunde PK, Holt E, Tonnessen T, Christensen G, Sejersted OM. 1998. Reduced myocardial Na+/ K+ pump capacity in congestive heart failure following myocardial infarction in rats. J Mol Cell Cardiol 30:1311–1328

Silverman B.Z., Fuller W, Eatona P, Deng J, Moormanc J. R,. Cheung J.Y, Jamese A.F, Shattock M. J. 2005. Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase. Cardiovascular Research 65: 93 – 103.

Stangherlin A, Zaccolo M. 2012. Phosphodiesterases and subcellular compartmentalized cAMP signaling in the cardiovascular system. Am J Physiol Heart Circ Physiol. 302(2):379-390

Stangherlin, A. et al. 2011. cGMP signals modulate cAMP levels in a compartmentspeciﬁc manner to regulate catecholamine-dependent signaling in cardiac myocytes. Circ. Res. 108, 929–939 Steinberg SF, Brunton LL. 2001. Compartmentation of G protein-coupled signaling pathways in cardiac myocytes. Annual Review of Pharmacology and Toxicology. 41:751–773.

Steiner AL, Kipnis DM, Utiger R, Parker C. 1969. Radioimmunoassay for the measurement of adenosine 3',5'-cyclic phosphate. Proc Natl Acad Sci U S A 64:367-73

Surdo N.C, Berrera M, Koschinski A, Brescia M, Machado M.R, Carr C, Wright P, Gorelik J, Morotti S, Grandi E, Bers D.M, Pantano S, Zaccolo M. 2017. FRET biosensor uncovers cAMP nano-domains at b-adrenergic targets that dictate precise tuning of cardiac contractility. Nat. Commun. 8, 15031.

Sweadner KJ and Rael E. 2000. The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression. Genomics 68: 41–56. Taylor S.S., Kim C., Cheng C.Y., Brown S.H.J., Wu J., Kannan N. 2008. Signaling through cAMP and cAMP-dependent protein kinase: Diverse strategies for drug design. Biochim. Biophys. Acta Proteins Proteom.1784:16–26.

(25)

Chapt

er

1

Taylor SS, Knighton DR, Zheng J, Ten Eyck LF, Sowadski JM. 1992. Structural framework for the protein kinase family. Annu Rev Cell Biol 8:429-62

Therien AG and Blostein R. 2000. Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 279: C541–C566.

Tulloch LB, Howie J, Wypijewski KJ, Wilson CR, Bernard WG, Shattock MJ, et al. 2011. The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation. J Biol Chem 286:36020–31.

Virshup D.M and Shenolikar S. 2009. From promiscuity to precision: protein phosphatases get a a makeover. Mol Cell. 33(5):537-45

Walaas SI, Czernik AJ, Olstad OK, Sletten K, and Walaas O. 1994. Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate phospholemman, an insulin and adrenaline-regulated membrane phosphoprotein,at specific sites in the carboxy terminal domain. Biochem J 304: 635– 640.

Wang J, Zhang XQ, Ahlers BA, Carl LL, Song J, Rothblum LI, Stahl RC,Carey DJ, Cheung JY. 2006. Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+exchanger. J Biol Chem.;281:32004–32014.

Zaccolo M, Pozzan T. 2000. Imaging signal transduction in living cells with GFP-based probes. IUBMB Life 49: 375–379.

Zaccolo M, Pozzan T. 2002. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295:1711–1715.

Zhang J, Hupfeld CJ, Taylor SS, Olefsky JM, Tsien RY. 2005. Insulin disrupts beta-adrenergic signaling to protein kinase A in adipocytes. Nature. 437(7058):569-73.

Zhang J, Ma Y, Taylor SS, Tsien RY. 2001. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A 98:14997-5002

Zhang XQ, Ahlers BA, Tucker AL, Song J, Wang J, Moorman JR, Mounsey JP, Carl LL, Rothblum LI, Cheung JY. 2006. Phospholemman inhibition of the cardiac Na+/Ca2+exchanger. Role of phosphorylation. J Biol Chem 281:7784–7792.