Exploration of abnormal dephosphorylation mechanism via Protein phosphatase 1β in heart failure

我们以前曾报道，蛋白磷酸酶1(PP 1)的过度激活是导致心力衰竭时磷蛋白(PLN)磷酸化减少以及伴随而来的心肌收缩能力降低的原因。在这一点上，PP 1β的表达水平在衰竭的心肌病仓鼠心脏中明显增加。然而，每种PP1亚型在心肌细胞中的具体作用尚不清楚。因此，我们用腺病毒RNA干扰技术研究了PP 1在收缩和磷酸化调节中的作用。方法：我们构建了编码短发夹状RNA的重组腺病毒，在8-10周龄的正常成年大鼠心肌细胞中诱导PP 1 a(Ad.PP1AI)、1β(Ad.PP1βI)和γ(Ad.PP1γI)的异构体特异性RNA干扰。以MOI分别为20、100、500的腺病毒载体分别转染分离的心肌细胞，72h后进行磷酸化、细胞缩短和细胞内钙瞬变的分析。结果：所有重组腺病毒RNAi表达均下降约90%，蛋白水平下降60%~80%，而对其他亚型表达无明显影响。磷蛋白(PLN)、A蛋白(PKA)、肌浆网ATPase(SERCA2a)、兰尼定受体2型(RyR2)等主要调控蛋白的表达水平未受影响。Ad.PP1Ai(1.5倍)和Ad.PP1βI(2倍)中PLN在Ser16位的磷酸化水平增加，但在PP1γ转染组中未见明显变化。在正常培养状态和β-肾上腺素能刺激状态下，PP 113I最有效地促进细胞缩短和钙瞬变。结论：Pp1βI可能是调节心肌细胞收缩的主要磷脂酶N磷酸酶.由于PP 113在各种心力衰竭模型和人类心力衰竭中的表达增加，因此它可能是心力衰竭的良好治疗靶点。

We previously reported that overactivation of protein phosphatase 1 (PP 1) is responsible for the decrease in phospholamban (PLN) phosphorylation and concomitant decrease in cardiac contractility in the failing heart. In this regard, increased expression level in PP 1β was evident in failing cardiomyopathic hamster hearts. However, specific role of each PP 1 isoform in cardiomyocytes is unclear. We therefore investigated the isoform specific role of PP 1 in contractility and phosphorylation regulation using adenoviral RNA interference (RNAi) technique.Methods : We created recombinant adenoviruses encoding short hairpin RNA, that induce isoform-specific RNAi for PP 1 a (Ad.PP1ai), 1β (Ad.PP1βi), and γ (Ad.PP1γi) in normal adult rat cardiomyocytes at 8-10 weeks of age. Isolated cardiomyocytes were transfected with each adenoviral vector at MOI of 20, 100, and 500, followed by the analysis for phosphorylation, cell shortening and intracellular calcium transient 72 hours after transfection. Results : All adenoviral RNAi transfection induced approximately 90% decrease in mRNA expression and significant decrease in protein level ranging from 60 to 80% without affecting other isoform expressions. Expression levels of major regulatory protein including phospholamban (PLN), A kinase (PKA), sarcoendoplasmic reticulum ATPase (SERCA2a), ryanodine receptor type 2 (RyR2) were not affected. Increase in PLN phosphorylation at Ser16 was observed in Ad.PP1ai (1.5 folds) and Ad.PP1βi (2 folds), but not in PP1γ-transfected cardiomyocytes. PP 113i most effectively augmented cell shortening and Ca^<2+> transient among the group at normal culture state, and under the beta-adrenergic stimulated state. Conclusion: PP 1βi appears to be the major PLN phosphatase that regulates cardiomyocyte contractility. Because PP 113 expression is increased in various heart failure models and human heart failure, it may be a good therapeutic target for heart failure.