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Real time cAMP dynamics in the vicinity of phospholemman in healthy and failing cardiomyocytes

dc.contributor.advisorNikolaev, Viacheslav Prof. Dr.
dc.contributor.authorBastug-Özel, Zeynep
dc.titleReal time cAMP dynamics in the vicinity of phospholemman in healthy and failing cardiomyocytesde
dc.contributor.refereeStühmer, Walter Prof. Dr.
dc.description.abstractengThe ubiquitous second messenger 3',5'-cyclic adenosine monophosphate (cAMP) is a crucial regulator of cardiac function and disease. It is known that cAMP signaling is mediated by discrete functional microdomains each containing a specific subset of differentially localized receptors, protein kinases and specific phosphodiesterases (PDEs). Cardiac phosphodiesterases (PDE1-5; PDE8 and 9) hydrolyze cyclic nucleotides, each with different selectivity and affinity for their substrates cAMP and 3',5'-cyclic guanosine monophosphate (cGMP). In one of these microdomains, the phosphorylation of the FXYD1 protein called Phospholemman (PLM), the negative regulator of Na+/K+-ATPase (NKA), via PKA or PKC leads to an increase of NKA activity and its sodium affinity, thereby lowering intracellular sodium levels and hence limiting cardiac inotropy. By obstructing high sodium levels during adrenergic stimulation it simultaneously favors calcium extrusion via the sodium-calcium exchanger (NCX). This mechanism may prevent calcium-overload, hypertrophy and triggered arrhythmias during cardiac stress. Interestingly, PLM expression is known to be altered in cardiomyocytes from postinfarcted rat hearts which results in depressed NKA activity. However, knowledge about the dynamics of cAMP pool coupled to PLM phosphorylation and the interactions of β-adrenergic receptors (β-AR) with individual cardiac PDE subtypes forming this important microdomain as well as their alterations in cardiac disease is insufficient. To investigate these questions, we developed a Förster Resonance Energy Transfer (FRET)-based PLM targeted cAMP biosensor PLM-E1. It can be used to precisely measure cAMP dynamics and to understand the regulatory mechanisms behind a possible local restriction of β-AR mediated cAMP signals in the PLM/NKA microdomain of adult rat ventricular myocytes (ARVMs). Using functional 86Rubidium-flux measurements in a PLM-E1 expressing stable HEK cell line, co-immunoprecipitation analysis of PLM-E1 transfected ARVMs and confocal microscopy, we showed that the newly developed PLM-E1 biosensor is associated with the α1 subunit (SU) of the NKA. The obtained FRET results were compared with measurements in the bulk cytosol performed using the cytosolic E1-camps sensor. It is well known that PDE2 activity constitutes a relatively low proportion of total cardiac PDE activity in the rat heart. However, our findings suggest that the actions of PDE2 regulate cAMP signals in the PLM microdomain in a compartmentalized manner at basal state (without prestimulation with β-AR agonists). Using the targeted FRET biosensor PLM-E1 in ARVMs we further analyzed the subtype specific β-AR control of cAMP in the PLM/NKA domain. Interestingly, the β2-AR showed a distinct control over the PLM microdomain during adrenergic stimulation. Local cAMP-FRET responses to PDE3 inhibition were well detectable in the PLM/NKA microdomain and showed a significant effect of this PDE in confining β2-AR signals to the vicinity of PLM upon adrenergic stimulation. Focusing on β2-AR stimulated cAMP pools in the PLM/NKA microdomain, we observed alterations of FRET responses in ARVMs from animals with chronic heart failure induced by myocardial infarction. These failing cells showed a significant loss of the PDE3 responses upon β2-AR stimulation and an almost compensating increase in PDE2 dependent control of cAMP in the vicinity of PLM. Strikingly, in this disease model the overall response of β2-AR to adrenergic stimulation in the PLM microdomain was reduced to a level comparable to the bulk cytosol. In this study, the efficiency of the targeted PLM-E1 biosensor and its potential for real time monitoring of compartmentalized cAMP signaling in adult rat ventricular myocytes was successfully demonstrated. In addition, the practicability of the tagged biosensor in ARVMs in a cardiac disease model was confirmed and analyzed. In particular, our data show that real time dynamics of cAMP in the PLM/NKA microdomain are significantly different from cytosolic cAMP in terms of local PDE regulation and direct receptor-mediated control of the microdomain. In heart failure, these mechanisms are seriously altered which might explain impaired regulation of PLM in disease, bringing out a potential therapeutic
dc.contributor.coRefereeSchwappach, Blanche Prof. Dr.
dc.subject.engSodium Potassium Pumpde
dc.affiliation.instituteMedizinische Fakultät
dc.subject.gokfullMedizin (PPN619874732)de

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