Fechner H, Suckau L, Kurreck J, Sipo I, Wang X, Pinkert S, Loschen S, Rekittke J, Weger S, Dekkers D, Vetter R, Erdmann V A, Schultheiss H-P, Paul M, Lamers J, Poller W
Department of Cardiology and Pneumology, Charité Universitätsmedizin Berlin, Berlin, Germany.
Gene Ther. 2007 Feb;14(3):211-8. doi: 10.1038/sj.gt.3302872. Epub 2006 Oct 5.
Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum Ca(2+) pump (SERCA2a) contributes to cardiac dysfunction in heart failure (HF). PLB downregulation may increase SERCA2a activity and improve cardiac function. Small interfering (si)RNAs mediate efficient gene silencing by RNA interference (RNAi). However, their use for in vivo gene therapy is limited by siRNA instability in plasma and tissues, and by low siRNA transfer rates into target cells. To address these problems, we developed an adenoviral vector (AdV) transcribing short hairpin (sh)RNAs against rat PLB and evaluated its potential to silence the PLB gene and to modulate SERCA2a-mediated Ca(2+) sequestration in primary neonatal rat cardiomyocytes (PNCMs). Over a period of 13 days, vector transduction resulted in stable > 99.9% ablation of PLB-mRNA at a multiplicity of infection of 100. PLB protein gradually decreased until day 7 (7+/-2% left), whereas SERCA, Na(+)/Ca(2+) exchanger (NCX1), calsequestrin and troponin I protein remained unchanged. PLB silencing was associated with a marked increase in ATP-dependent oxalate-supported Ca(2+) uptake at 0.34 microM of free Ca(2+), and rapid loss of responsiveness to protein kinase A-dependent stimulation of Ca(2+) uptake was maintained until day 7. In summary, these results indicate that AdV-derived PLB-shRNA mediates highly efficient, specific and stable PLB gene silencing and modulation of active Ca(2+) sequestration in PNCMs. The availability of the new vector now enables employment of RNAi for the treatment of HF in vivo.
受磷蛋白（PLB）调节的肌浆网Ca²⁺泵（SERCA2a）功能受损会导致心力衰竭（HF）中的心脏功能障碍。PLB下调可能会增加SERCA2a活性并改善心脏功能。小干扰（si）RNA通过RNA干扰（RNAi）介导有效的基因沉默。然而，它们在体内基因治疗中的应用受到血浆和组织中siRNA不稳定性以及siRNA向靶细胞转移率低的限制。为了解决这些问题，我们开发了一种腺病毒载体（AdV），其转录针对大鼠PLB的短发夹（sh）RNA，并评估了其在原代新生大鼠心肌细胞（PNCMs）中沉默PLB基因以及调节SERCA2a介导的Ca²⁺摄取的潜力。在13天的时间里，以100的感染复数进行载体转导导致PLB - mRNA稳定地> 99.9%缺失。PLB蛋白逐渐下降直至第7天（剩余7±2%），而SERCA、钠/钙交换体（NCX1）、肌集钙蛋白和肌钙蛋白I蛋白保持不变。在游离Ca²⁺浓度为0.34微摩尔时，PLB沉默与ATP依赖的草酸盐支持的Ca²⁺摄取显著增加相关，并且对蛋白激酶A依赖的Ca²⁺摄取刺激的快速反应丧失一直维持到第7天。总之，这些结果表明，AdV衍生的PLB - shRNA介导了高效、特异且稳定的PLB基因沉默以及PNCMs中活性Ca²⁺摄取的调节。这种新载体的可用性现在使得能够在体内将RNAi用于HF的治疗。
