Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes.
Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes.
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DOI：
10.1093/cvr/cvu029
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发表时间：
2014-04
影响因子：
10.8
通讯作者：
Zeineb Haj Slimane;I. Bedioune;P. Lechêne;A. Varin;F. Lefebvre;P. Mateo;V. Domergue‐Dupont;M. Dewenter;Wito Richter;M. Conti;A. El-Armouche;Jin Zhang;R. Fischmeister;G. Vandecasteele
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作者：
Zeineb Haj Slimane;I. Bedioune;P. Lechêne;A. Varin;F. Lefebvre;P. Mateo;V. Domergue‐Dupont;M. Dewenter;Wito Richter;M. Conti;A. El-Armouche;Jin Zhang;R. Fischmeister;G. Vandecasteele
camp依赖性蛋白激酶（PKA）介导β-肾上腺素能受体（β-AR）对心脏收缩和基因表达的调控。尽管PKA活性在成人心肌细胞的各种亚细胞区室中都有很好的特征，但其在细胞核中的调控在很大程度上仍然未知。本研究的目的是比较PKA在心肌细胞细胞质和细胞核中的调节方式。方法与结果采用靶向荧光共振能量转移探针测定成年大鼠心室肌细胞胞浆和细胞核cAMP、PKA活性。异丙肾上腺素（Iso）刺激β-AR导致两个室室cAMP快速升高，而PKA活性在细胞质中快速升高，但在细胞核中明显较慢。Iso对细胞质的激活也比核PKA更有效。L-858051激活腺苷酸环化酶或3-异丁基-1-甲基xantine抑制磷酸二酯酶（PDE）时，核PKA激活的缓慢动力学类似。与此一致的是，Iso （15 s）脉冲刺激最大限度地诱导了细胞质中PKA和肌球蛋白结合蛋白C的磷酸化，但在细胞核中轻微激活了PKA和cAMP反应元件结合蛋白的磷酸化。PDE4的抑制或Pde4d基因的消融延长了小鼠细胞质PKA的激活并增强了核PKA反应。在细胞质中，磷酸酶1 （PP1）和磷酸酶2A （PP2A）参与了PKA反应的终止，而在细胞核中只有磷酸酶1起作用。结论：我们的研究揭示了心肌细胞对β-AR刺激的细胞质和核PKA反应的差异整合。这可能对β-AR刺激的生理和病理肥厚反应具有重要意义。
AIMS The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-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. β-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 β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation.