Evidence for protein phosphatase inhibitor-1 playing an amplifier role in β-adrenergic signaling in cardiac myocytes
Evidence for protein phosphatase inhibitor-1 playing an amplifier role in β-adrenergic signaling in cardiac myocytes
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DOI：
10.1096/fj.02-0057fje
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发表时间：
2003-01-01
期刊：
影响因子：
4.8
通讯作者：
Eschenhagen, T
中科院分区：
文献类型：
--
作者：
El-Armouche, A;Rau, T;Eschenhagen, T
蛋白磷酸酶抑制剂-1 （PPI-1）仅在被蛋白激酶A磷酸化时才能抑制1型磷酸酶（PP1），并在磷酸化/去磷酸化平衡中发挥关键作用。通过逆转录聚合酶链反应克隆大鼠心脏PPI-1，在大肠杆菌中表达，在磷酸酶试验中评估，并用于生成抗血清。在不同的巨细胞病毒启动子（AdPPI-1/GFP）下构建了一种编码PPI-1和绿色荧光蛋白（GFP）的腺病毒。仅用gfp病毒（AdGFP）作为对照。以新生大鼠心肌细胞工程心脏组织（EHT）和成年大鼠心肌细胞（ARCMs）为模型系统。采用Northern blots法测定人心室样品中PPI-1的表达。与AdGFP相比，AdPPI-1/ gfp感染的新生大鼠心肌细胞PP1活性降低73%。感染AdPPI-1/GFP的eht对异丙肾上腺素的敏感性增加了5倍。当用1nm异丙肾上腺素刺激AdPPI-1/ gfp感染的ARCMs时，细胞缩短和磷酸化增强。与非衰竭心脏（n=8）相比，伴有扩张性心肌病和缺血性心肌病的衰竭心脏（n=8）的PPI-1 mRNA水平降低了57+/-12%。总之，增加的PPI-1表达增强了肌细胞对异丙肾上腺素的敏感性，表明PPI-1在β -肾上腺素能信号传导中起放大作用。衰竭心脏PPI-1的降低可能参与cAMP通路的脱敏。
The protein phosphatase inhibitor-1 (PPI-1) inhibits phosphatase type-1 (PP1) only when phosphorylated by protein kinase A and could play a pivotal role in the phosphorylation/dephosphorylation balance. Rat cardiac PPI-1 was cloned by reverse transcriptase-polymerase chain reaction, expressed in Eschericia coli, evaluated in phosphatase assays, and used to generate an antiserum. An adenovirus was constructed encoding PPI-1 and green fluorescent protein (GFP) under separate cytomegalovirus promotors (AdPPI-1/GFP). A GFP-only virus (AdGFP) served as control. Engineered heart tissue (EHT) from neonatal rat cardiomyocytes and adult rat cardiac myocytes (ARCMs) were used as model systems. PPI-1 expression was determined in human ventricular samples by Northern blots. Compared with AdGFP, AdPPI-1/GFP-infected neonatal rat cardiomyocytes displayed a 73% reduction in PP1 activity. EHTs infected with AdPPI-1/GFP exhibited a fivefold increase in isoprenaline sensitivity. AdPPI-1/GFP-infected ARCMs displayed enhanced cell shortening as well as enhanced phospholamban phosphorylation when stimulated with 1 nM isoprenaline. PPI-1 mRNA levels were reduced by 57+/-12% in failing hearts with dilated and ischemic cardiomyopathy (n=8 each) compared with nonfailing hearts (n=8). In summary, increased PPI-1 expression enhances myocyte sensitivity to isoprenaline, indicating that PPI-1 acts as an amplifier in beta-adrenergic signaling. Decreased PPI-1 in failing human hearts could participate in desensitization of the cAMP pathway.