6 research outputs found
Interventricular Differences in ÎČâAdrenergic Responses in the Canine Heart: Role of Phosphodiesterases
Background
RV and LV have different embryologic, structural, metabolic, and electrophysiologic characteristics, but whether interventricular differences exist in ÎČâadrenergic (ÎČâAR) responsiveness is unknown. In this study, we examine whether ÎČâAR response and signaling differ in right (RV) versus left (LV) ventricles.
Methods and Results
Sarcomere shortening, Ca2+ transients, ICa,L and IKs currents were recorded in isolated dog LV and RV midmyocytes. Intracellular [cAMP] and PKA activity were measured by live cell imaging using FRETâbased sensors. Isoproterenol increased sarcomere shortening â10âfold and Ca2+âtransient amplitude â2âfold in LV midmyocytes (LVMs) versus â25âfold and â3âfold in RVMs. FRET imaging using targeted Epac2camps sensors revealed no change in subsarcolemmal [cAMP], but a 2âfold higher ÎČâAR stimulation of cytoplasmic [cAMP] in RVMs versus LVMs. Accordingly, ÎČâAR regulation of ICa,L and IKs were similar between LVMs and RVMs, whereas cytoplasmic PKA activity was increased in RVMs. Both PDE3 and PDE4 contributed to the ÎČâAR regulation of cytoplasmic [cAMP], and the difference between LVMs and RVMs was abolished by PDE3 inhibition and attenuated by PDE4 inhibition. Finally LV and RV intracavitary pressures were recorded in anesthetized beagle dogs. A bolus injection of isoproterenol increased RV dP/dtmaxâ5âfold versus 3âfold in LV.
Conclusion
Canine RV and LV differ in their ÎČâAR response due to intrinsic differences in myocyte ÎČâAR downstream signaling. Enhanced ÎČâAR responsiveness of the RV results from higher cAMP elevation in the cytoplasm, due to a decreased degradation by PDE3 and PDE4 in the RV compared to the LV
Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes.
Dynamique Spatiotemporelle de la protéine kinase AMPc dépendante dans les myocytes cardiaques
The cAMP-dependent protein kinase (PKA) exerts short term beneficial effects on cardiac function by phosphorylating several key excitation-contraction coupling (ECC) proteins. However, its chronic activation is deleterious on the long term, and this may involve regulation of nuclear effectors ultimately leading to hypertrophic remodelling and heart failure. The subcellular localization of PKA, mediated by anchoring proteins (AKAPs), is important for the speed and specificity of hormones that activate the cAMP pathway. The levels of cAMP are regulated by adenylyl cyclase and phosphodiesterases (PDEs), and PKA activity is counterbalanced by Ser/Thr phosphatases (PPs). In heart, the most important PDEs that degrade cAMP belong to the PDE3 and PDE4 famillies, whereas the major cardiac PPs are PP1, PP2A and PP2B. In a first part, I developed, in adult rat cardiomyocytes, a technique to measure PKA activity in real time specifically in the cytoplasm and the nucleus. For this I used genetically-encoded fluorescence resonance energy transfer (FRET) sensors called AKAR (A-kinase activity reporters) that can be targeted specifically to the nucleus or the cytoplasm by nuclear localization or exclusion sequences, respectively. Using this approach, I showed that maintained ÎČ-adrenergic stimulation activates PKA more efficiently and more potently in the cytoplasm than in the nucleus, and that the kinetics of PKA activation was much slower in the nucleus than in the cytoplasm. Accordingly, a short ÎČ-adrenergic stimulation maximally activated PKA in the cytoplasm but marginally activated PKA in the nucleus. In a second part, I characterized the respective contribution of PDE3, PDE4, and PP1, PP2A and PP2B families in the regulation of cytoplasmic and nuclear PKA activity in response to ÎČ-adrenergic stimulation. PDE4, but not PDE3, regulates PKA activity in the cytoplasm and in the nucleus. The use of knock out mice for Pde4b and Pde4d genes revealed that PDE4B plays a predominant role to modulate ÎČ-AR stimulation of cytoplasmic PKA, whereas in the nucleus both PDE4B and PDE4D isoforms contribute. Finally, I showed that both PP1 and PP2A, but not PP2B, participate to the termination of ÎČ-adrenergic PKA responses in the cytoplasm, whereas PP1 appears to play a major role in the nuclei. In conclusion, this work highlights the role of phosphodiesterases and phosphatases in the differential integration of PKA responses to ÎČ-adrenergic stimulation in the cytoplasm and the nucleus of adult cardiomyocytes.La protĂ©ine kinase AMPc-dĂ©pendante (PKA) joue un rĂŽle crucial dans la rĂ©gulation neurohormonale de la fonction cardiaque. Lâactivation aiguĂ« de la PKA est bĂ©nĂ©fique car elle conduit Ă une augmentation de la contraction cardiaque en phosphorylant les acteurs clĂ©s du couplage excitation-contraction. En revanche, son activation chronique est dĂ©lĂ©tĂšre et ces effets semblent faire intervenir la rĂ©gulation de protĂ©ines nuclĂ©aires pouvant conduire au remodelage hypertrophique et Ă l'insuffisance cardiaque. La localisation subcellulaire de la PKA, assurĂ©e par des protĂ©ines dâancrage (AKAPs), est importante pour la rapiditĂ© et la spĂ©cificitĂ© dâaction des hormones mettant en jeu la voie de lâAMPc. Les niveaux dâAMPc sont rĂ©gulĂ©s par lâactivitĂ© des adĂ©nylate cyclases et des phosphodiestĂ©rases (PDEs), et lâĂ©tat de phosphorylation des protĂ©ines cibles de la PKA dĂ©pend de lâactivitĂ© des Ser/Thr phosphatases (PPs). Dans le cĆur, les PDEs les plus importantes dĂ©gradant lâAMPc sont les PDE3 et les PDE4. Les principales PPs cardiaques sont PP1, PP2A et PP2B. Dans une premiĂšre partie de mon travail, jâai mis au point, dans les cardiomyocytes de rats adultes, une mesure de lâactivitĂ© de la PKA en temps rĂ©el dans les compartiments cytoplasmiques et nuclĂ©aires. Jâai utilisĂ© pour cela des sondes de type AKAR (A-kinase activity reporters) basĂ©es sur le transfert dâĂ©nergie de fluorescence (FRET) et localisĂ©es spĂ©cifiquement dans le noyau ou dans le cytoplasme par des sĂ©quences dâadressage ou dâexclusion nuclĂ©aires. Jâai ainsi pu montrer quâune stimulation maintenue des rĂ©cepteurs ÎČ-adrĂ©nergiques active la PKA de façon plus importante dans le cytoplasme que dans le noyau, et que cette activation se dĂ©veloppe lentement au niveau nuclĂ©aire que dans le cytoplasme. De ce fait, une stimulation brĂšve des rĂ©cepteurs ÎČ-adrĂ©nergiques active maximalement la PKA dans le cytoplasme, mais de façon marginale dans le noyau. Dans une seconde partie de lâĂ©tude, je me suis intĂ©ressĂ©e au rĂŽle des PDE3 et PDE4 ainsi quâĂ celui de PP1, PP2A et PP2B dans la rĂ©gulation de lâactivitĂ© PKA cytoplasmique et nuclĂ©aire, en rĂ©ponse Ă une stimulation ÎČ-adrĂ©nergique. Jâai montrĂ© que la PDE4, mais pas la PDE3, rĂ©gule lâactivitĂ© de la PKA cytoplasmique et nuclĂ©aire. Lâutilisation de souris invalidĂ©es pour les gĂšnes Pde4b et Pde4d a rĂ©vĂ©lĂ© que lâisoforme PDE4B est prĂ©dominante pour la modulation de lâactivitĂ© PKA cytoplasmique, alors que les deux isoformes PDE4B et PDE4D contribuent Ă la rĂ©gulation de lâactivitĂ© PKA nuclĂ©aire. Finalement, jâai montrĂ© que la PP1 et la PP2A, mais pas la PP2B, participent Ă la terminaison des rĂ©ponses ÎČ-adrĂ©nergiques dans le cytoplasme, alors quâau niveau nuclĂ©aire, la PP1 semble jouer un rĂŽle majeur. En conclusion, ce travail a mis en Ă©vidence le rĂŽle des phosphodiestĂ©rases et des phosphatases dans lâintĂ©gration diffĂ©rentielle des rĂ©ponses PKA Ă une stimulation ÎČ-adrĂ©nergique dans le cytoplasme et le noyau de cardiomyocytes adultes
Spatiotemporal dynamic of cAMP-dependent protein kinase in cardiac myocytes
La protĂ©ine kinase AMPc-dĂ©pendante (PKA) joue un rĂŽle crucial dans la rĂ©gulation neurohormonale de la fonction cardiaque. Lâactivation aiguĂ« de la PKA est bĂ©nĂ©fique car elle conduit Ă une augmentation de la contraction cardiaque en phosphorylant les acteurs clĂ©s du couplage excitation-contraction. En revanche, son activation chronique est dĂ©lĂ©tĂšre et ces effets semblent faire intervenir la rĂ©gulation de protĂ©ines nuclĂ©aires pouvant conduire au remodelage hypertrophique et Ă l'insuffisance cardiaque. La localisation subcellulaire de la PKA, assurĂ©e par des protĂ©ines dâancrage (AKAPs), est importante pour la rapiditĂ© et la spĂ©cificitĂ© dâaction des hormones mettant en jeu la voie de lâAMPc. Les niveaux dâAMPc sont rĂ©gulĂ©s par lâactivitĂ© des adĂ©nylate cyclases et des phosphodiestĂ©rases (PDEs), et lâĂ©tat de phosphorylation des protĂ©ines cibles de la PKA dĂ©pend de lâactivitĂ© des Ser/Thr phosphatases (PPs). Dans le cĆur, les PDEs les plus importantes dĂ©gradant lâAMPc sont les PDE3 et les PDE4. Les principales PPs cardiaques sont PP1, PP2A et PP2B. Dans une premiĂšre partie de mon travail, jâai mis au point, dans les cardiomyocytes de rats adultes, une mesure de lâactivitĂ© de la PKA en temps rĂ©el dans les compartiments cytoplasmiques et nuclĂ©aires. Jâai utilisĂ© pour cela des sondes de type AKAR (A-kinase activity reporters) basĂ©es sur le transfert dâĂ©nergie de fluorescence (FRET) et localisĂ©es spĂ©cifiquement dans le noyau ou dans le cytoplasme par des sĂ©quences dâadressage ou dâexclusion nuclĂ©aires. Jâai ainsi pu montrer quâune stimulation maintenue des rĂ©cepteurs ÎČ-adrĂ©nergiques active la PKA de façon plus importante dans le cytoplasme que dans le noyau, et que cette activation se dĂ©veloppe lentement au niveau nuclĂ©aire que dans le cytoplasme. De ce fait, une stimulation brĂšve des rĂ©cepteurs ÎČ-adrĂ©nergiques active maximalement la PKA dans le cytoplasme, mais de façon marginale dans le noyau. Dans une seconde partie de lâĂ©tude, je me suis intĂ©ressĂ©e au rĂŽle des PDE3 et PDE4 ainsi quâĂ celui de PP1, PP2A et PP2B dans la rĂ©gulation de lâactivitĂ© PKA cytoplasmique et nuclĂ©aire, en rĂ©ponse Ă une stimulation ÎČ-adrĂ©nergique. Jâai montrĂ© que la PDE4, mais pas la PDE3, rĂ©gule lâactivitĂ© de la PKA cytoplasmique et nuclĂ©aire. Lâutilisation de souris invalidĂ©es pour les gĂšnes Pde4b et Pde4d a rĂ©vĂ©lĂ© que lâisoforme PDE4B est prĂ©dominante pour la modulation de lâactivitĂ© PKA cytoplasmique, alors que les deux isoformes PDE4B et PDE4D contribuent Ă la rĂ©gulation de lâactivitĂ© PKA nuclĂ©aire. Finalement, jâai montrĂ© que la PP1 et la PP2A, mais pas la PP2B, participent Ă la terminaison des rĂ©ponses ÎČ-adrĂ©nergiques dans le cytoplasme, alors quâau niveau nuclĂ©aire, la PP1 semble jouer un rĂŽle majeur. En conclusion, ce travail a mis en Ă©vidence le rĂŽle des phosphodiestĂ©rases et des phosphatases dans lâintĂ©gration diffĂ©rentielle des rĂ©ponses PKA Ă une stimulation ÎČ-adrĂ©nergique dans le cytoplasme et le noyau de cardiomyocytes adultes.The cAMP-dependent protein kinase (PKA) exerts short term beneficial effects on cardiac function by phosphorylating several key excitation-contraction coupling (ECC) proteins. However, its chronic activation is deleterious on the long term, and this may involve regulation of nuclear effectors ultimately leading to hypertrophic remodelling and heart failure. The subcellular localization of PKA, mediated by anchoring proteins (AKAPs), is important for the speed and specificity of hormones that activate the cAMP pathway. The levels of cAMP are regulated by adenylyl cyclase and phosphodiesterases (PDEs), and PKA activity is counterbalanced by Ser/Thr phosphatases (PPs). In heart, the most important PDEs that degrade cAMP belong to the PDE3 and PDE4 famillies, whereas the major cardiac PPs are PP1, PP2A and PP2B. In a first part, I developed, in adult rat cardiomyocytes, a technique to measure PKA activity in real time specifically in the cytoplasm and the nucleus. For this I used genetically-encoded fluorescence resonance energy transfer (FRET) sensors called AKAR (A-kinase activity reporters) that can be targeted specifically to the nucleus or the cytoplasm by nuclear localization or exclusion sequences, respectively. Using this approach, I showed that maintained ÎČ-adrenergic stimulation activates PKA more efficiently and more potently in the cytoplasm than in the nucleus, and that the kinetics of PKA activation was much slower in the nucleus than in the cytoplasm. Accordingly, a short ÎČ-adrenergic stimulation maximally activated PKA in the cytoplasm but marginally activated PKA in the nucleus. In a second part, I characterized the respective contribution of PDE3, PDE4, and PP1, PP2A and PP2B families in the regulation of cytoplasmic and nuclear PKA activity in response to ÎČ-adrenergic stimulation. PDE4, but not PDE3, regulates PKA activity in the cytoplasm and in the nucleus. The use of knock out mice for Pde4b and Pde4d genes revealed that PDE4B plays a predominant role to modulate ÎČ-AR stimulation of cytoplasmic PKA, whereas in the nucleus both PDE4B and PDE4D isoforms contribute. Finally, I showed that both PP1 and PP2A, but not PP2B, participate to the termination of ÎČ-adrenergic PKA responses in the cytoplasm, whereas PP1 appears to play a major role in the nuclei. In conclusion, this work highlights the role of phosphodiesterases and phosphatases in the differential integration of PKA responses to ÎČ-adrenergic stimulation in the cytoplasm and the nucleus of adult cardiomyocytes
Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes
AIMS: The cAMP-dependent protein kinase (PKA) mediates ÎČ-adrenoceptor (ÎČ-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. METHODS AND RESULTS: Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. ÎČ-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. CONCLUSION: Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to ÎČ-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to ÎČ-AR stimulation