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

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Understanding compartmentalized cAMP signaling for potential therapeutic approaches in

cardiac disease

Musheshe, Nshunge

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Publication date: 2018

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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.

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Chapter 5. General Discussion and Future

Perspectives

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GENERAL DISCUSSION

β-Adrenoceptors (β-AR) belong to a family of G-protein coupled receptors (GPCRs) at the plasmalemma. Ligand binding to β-AR enhances cyclic AMP (cAMP) levels and protein kinase A (PKA) activity required for catecholaminergic regulation of cardiac function. Hormone-induced cAMP/PKA signaling leads to phosphorylation of proteins involved in excitation-contraction coupling (ECC) which in some instances may result in opposing effects on [Ca2+_]

i. It has been illustrated that on adrenergic stimulation, PKA-mediated phosphorylation of L-type Ca2+_{channels (LTCC) and phospholamban (PLB)} leads to increased [Ca2+_]

i and positive inotropy, while PKA-mediated phosphorylation of Troponin I (TPNI) reduces the affinity of the myofilament for Ca2+ _{thereby possibly abolishing the effect of increased [Ca}2+_]

i. A similar outcome on [Ca2+_]

i is achieved via PKA-mediated phosphorylation of phospholemman (PLM) at Ser68, which results in enhanced Na+_/K+_{ATPase (NKA) activity.} Phosphorylated PLM releases its inhibition of the NKA pump resulting in the pump’s increased affinity for Na+_{(Silvermana, B.Z et al., 2005). The stimulated} NKA pumps Na+_{outside of the cell which in turn enhances extrusion of Ca}2+_via the Na+_/Ca2+_{Exchanger (NCX) and hence reduced [Ca}2+_]

i and inotropy. How β-adrenergic receptor stimulation coordinates these apparently opposing effects is unclear.

Compartmentalized signaling by the cAMP/PKA pathway, has been shown to be required to selectively activate distinct PKA subsets thereby ensuring hormone-specific responses within the same cell. cAMP and its effectors and regulators are spatially organized in distinct subcellular domains and only a fraction of all domains is activated in response to a given hormone. Dysfunction in such organization has been implicated in various cardiac diseases such as heart failure

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and arrhythmia (MacLenna, D.H and Kranias, E.G., 2003; Venetucci, L.A et al., 2008). A study by Surdo N.C et al., 2017 recently demonstrated that even downstream of a single hormone receptor, multiple cAMP pools can be generated, each showing distinct amplitude and kinetics of the cAMP signal. The study demonstrated that, on β-adrenergic receptor stimulation, distinct cAMP signals are generated at the LTCC/A Kinase Anchoring Protein 79(AKAP79) complex at the plasmalemma and at the myofilaments and that such heterogeneity is required for optimal regulation of inotropy. The overarching hypothesis of the current study was that a similar distinct handling of cAMP and PKA may occur at the PLM-NKA complex and LTCC/AKAP79 complex to coordinate the opposing effects on [Ca2+_]

i observed.

FRET-based sensors for cAMP and PKA activity studies allow for the demonstration that cAMP – PKA signaling pathway is compartmentalized. In

chapter 2, we review the evidence in support of cAMP nanodomains and how

advancements in FRET-based tools has made it possible to study cAMP and PKA activity in real time with high spatial and temporal resolution. Using FRET-based tools, it has been demonstrated that cAMP increase is not homogenous throughout the intact cell allowing for the manifestation of distinct responses downstream of hormonal stimulation (Zaccolo M and Pozzan T., 2002, Surdo N.C et al., 2017, Musheshe N et al., 2018). This tight spatial regulation is achieved in part by PDEs which degrade cAMP and prevent its diffusion between compartments and prevent unnecessary PKA activation (Di Benedetto G et al., 2008; Mika D et al., 2012), and by AKAPs which bind and target PKA to specific sites. Phosphatases also play a role in compartmentalization by dephosphorylating PKA targets and thus terminating the signal. cAMP nanodomains are defined therefore by unique cAMP pools, PDEs, PKA and its

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regulators, AKAPs and phosphatases. Novel FRET-based sensors allow for the comparison of cAMP – PKA signals in different cAMP nanodomains in an intact cell.

In chapter 3, we investigated whether the PLM-NKA complex and

LTCC/AKAP79 are regulated by distinct cAMP and PKA signals which may contribute to the opposing effects on [Ca2+_]

I observed at the respective sites. In these studies, using targeted FRET-based sensors for cAMP in ARVMs and NRVMs, we revealed heterogeneity in the cAMP response on β-AR stimulation with cAMP response at PLM-NKA being significantly lower than that at LTCC/AKAP79. We determined that the differential increase in cAMP at PLM was β-adrenergic specific as global stimulation of ACs resulted in a sizeable increase in cAMP at both PLM and AKAP79. The heterogeneity in cAMP amplitudes was found to be due to PDEs with PDEs 2 and 8 found to significantly contribute to the attenuation of cAMP at PLM-NKA whereas they had no significant effect in the regulation of the cAMP response at LTCC/AKAP79. We also showed that despite revealing heterogenous increase in cAMP at PLM-NKA and LTCC/AKAP79 on β-AR stimulation, PKA dependent phosphorylation at the two sites is similar. Further investigation revealed a higher phosphatase activity at AKAP79 than at PLM. There is evidence from previous studies that suggests that phosphatase activity may differ substantially in different subcellular cardiac myocyte microdomains (Marx S.O et al., 2000, Yano M et al., 2005). As it is already established that protein phosphatase 1 (PP1) dephosphorylates PLM at the PKA site S68 (El-Armouche et al., 2011), and that AKAP79 interacts with protein phosphatase 2B (PP2B) and PP1 (Le A.V et al., 2011), these phosphatases are likely to be involved in the differential local regulation of PKA-dependent phosphorylation. Further studies however will be required to define the type of phosphatases involved and their relative contribution at the AKAP79/LTCC and

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PLM/NKA complexes. Additional studies will also be required to unravel the mechanism involved in the biphasic dephosphorylation that we observed at PLM-NKA and LTCC/AKAP79 complexes and its functional relevance at the respective sites. Overall, the finding presented in chapter 3 support a model whereby cAMP/PKA signaling is uniquely regulated at AKAP79/LTCC and PLM/NKA by PDEs and phosphatases to ensure compartmentalized signaling at the two sites and allow coordination of ion (Ca2+_{and Na}+_{) flux across the} plasmalemma that is essential for optimal regulation of cardiac contraction and relaxation.

It is crucial that targeted sensors genuinely report local signals as they occur in wildtype cells. Previous studies have shown that fusing of targeting domains to FRET-based sensors such as Epac-camps 1, affects its FRET performance due to steric hinderance of the FRET pair by the targeting domain (Surdo N.C et al., 2017), however there are no investigations so far to determine whether targeting domains may affect cAMP and PKA activity readouts by the FRET-based sensors at the respective sites. In chapter 4 therefore, we validated AKAP5-targeted sensors for cAMP and PKA activity studies. Our studies showed that the ability of AKAP5-targeted sensors to recruit PKA to AKAP5-specific nanodomains does not affect local cAMP and PKA activity readouts. These results confirm that our findings using these targeted reporters with AKAP5 as a targeting domain are accurate and do not include artifacts.

In conclusion, by using FRET-based tools, our findings reveal a novel facet of the complex regulation of local cAMP signaling in cardiac myocytes and support tight coordination of PKA activity at the plasmalemma to optimally modulate ion flux (Figure 1).

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Figure 1: Schematic representation of PKA-mediated phosphorylation of LTCC/AKAP79 and

PLM/NKA resulting in opposing effects on Ca2+_{. PDE is phosphodiesterase. PP is protein}

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FUTURE

PERSPECTIVES:

FRET-based sensors are critical for unraveling cAMP nanodomains and their unique signaling features. Future studies will require further adjusting the sensitivity of the sensors and improving their dynamic range in order to refine our understanding of cAMP-regulated cardiomyocyte compartments and provide further information on components that play an important role in normal physiology and development of heart disease. The discovery of inhibitors is an essential part of drug discovery (Maurice D.H et al., 2014). Not only can inhibitors be developed into therapeutic agents or diagnostics, they may also be used as tools to study biological or pathological processes. However, of note is that PDE inhibitors are chemical compounds which makes it most likely difficult to fuse them to FRET sensors which are protein-based. And in addition, application of inhibitors in patients may require encapsulation or use in form of inhalers (Lim G.B., 2018) to aid delivery in the body.

Both PDE2 (Hua R et al., 2012; Aye T.T., 2012) and PP1 (Neumann et al., 1997) have been shown to be upregulated both in human and experimental HF. The data we present suggests that in HF further attenuation of the cAMP response at PLM/NKA by increased PDE2 activity and enhanced PLM dephosphorylation by PP1 may concur to reduce the adrenergic regulation of NKA, potentially worsening elevated [Na+_]

i and aggravating the negative effects on cardiac metabolism and oxidative stress that are associated with HF. PDE2 inhibition therefore may positively influence cardiac performance.

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Various studies point to the relevance of PDE2 and its targeting in heart disease (Vettel C et al., 2017; Zoccarato A et al., 2015) and recently a study by Monterisi S et al., 2017 also demonstrated that inhibition of PDE2A2 results in elongated mitochondria and protection from mitochondrial dependent-cell death in cardiac myocytes. Despite a huge interest in pharmaceutical companies to develop novel PDE2 inhibitors besides those already available for pharmacological use, so far there is no PDE2 inhibitors for clinical use in the treatment of cardiac disorders such as heart failure, but a clinical trial in phase 1 is underway for the development of the PDE2A inhibitor for the treatment of Schizophrenia (Takeda., 2015) indicating possible application of PDE2 inhibitors in diseased conditions.

The exclusive increase in cAMP levels at the PLM/NKA complex, but not at the AKAP79/LTCC complex, on inhibition of PDE2 and PDE8 opens the possibility to target these enzymes by designing the respective novel chemical inhibitors to achieve selective manipulation of NKA activity for therapeutic purposes. Little information is known about the role of PDE8 in the heart. A study by Patrucco E et al., 2010 indicated that PDE8A plays a role in one or more pools of cAMP implicated in the regulation of ECC in ventricular myocytes PDE8 null myocytes had higher [Ca2+_]

I and Ica, than the wild type. However, our study is the first evidence of a specific signalosome i.e. PLM/NKA regulated by PDE8 in ventricular myocytes.

Our findings strongly point to the functional relevance of PDE2 and PDE8 (Figure 1) in the heart making their targeting critical candidates for treatment. In addition, understanding of the type of phosphatases involved in controlling PKA at the two sites also provides a platform for targeted therapies in diseased conditions.

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Future studies will require identifying the respective PDE2 and PDE8 isoforms to further encourage precise therapy and avoid adverse side effects that are observed in PDE inhibition (Maurice D.H et al., 2014). However, as discussed by Musheshe N et al., 2018- targeting of isoforms of each respective PDE family is most likely difficult to achieve as isoforms share the same characteristics in the catalytic site. Therefore, as opposed to inhibiting family-specific isoforms, use of peptides or small molecules that target the interaction of PDE isoforms with the signalosome may be more plausible as this would allow for the displacement of PDE isoforms from their signalosomes.

We hope that our work will open doors for new experimental strategies ideas on understanding local cAMP – PKA signaling at various nanodomains in the heart including but not limited to those involved in ECC.

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