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

我们之前报道过，蛋白磷酸酶1 （pp1）的过度激活是导致衰竭心脏中磷蛋白（PLN）磷酸化降低和伴随的心脏收缩力下降的原因。在这方面，PP 1β表达水平在心力衰竭的心肌病仓鼠心脏中明显升高。然而，每种pp1亚型在心肌细胞中的具体作用尚不清楚。因此，我们使用腺病毒RNA干扰（RNAi）技术研究了pp1在收缩性和磷酸化调控中的异构体特异性作用。方法：构建编码短发夹RNA的重组腺病毒，诱导pp1a （Ad）亚型特异性RNAi。PP1ai), 1β (Ad；PP1βi)和γ (Ad.PP1γi)在8-10周龄正常成年大鼠心肌细胞中的表达。分别在MOI为20,100和500时转染分离的心肌细胞，然后在转染72小时后分析磷酸化，细胞缩短和细胞内钙瞬态。结果：所有腺病毒RNAi转染诱导mRNA表达降低约90%，蛋白水平显著降低60 - 80%，但不影响其他异构体的表达。主要调节蛋白包括磷蛋白（PLN）、A激酶（PKA）、肌内质网atp酶（SERCA2a）、ryanodine受体2型（RyR2）的表达水平未受影响。在Ad中观察到PLN第16丝氨酸磷酸化增加。PP1ai（1.5倍）和Ad。PP1βi（2倍），但在pp1 γ-转染的心肌细胞中没有。在正常培养状态和β -肾上腺素能刺激状态下，pp113i最有效地增强了细胞缩短和Ca^<2+>瞬态。结论：pp1 βi可能是调节心肌细胞收缩性的主要PLN磷酸酶。由于PP 113在各种心力衰竭模型和人类心力衰竭中表达增加，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.