Phospholamban ablation by RNA interference increases Ca2+ uptake into rat cardiac myocyte sarcoplasmic reticulum

Phospholarnban (PLB) inhibits SR Ca2+-ATPase 2 (SERCA2) Ca2+ uptake and is a potential therapeutic target in the context of heart failure. RNA interference (RNAi) is a technique that produces sequence-specific, post-transcriptional gene silencing through the use of double-stranded RNA directed against the homologous target gene. The goal of the current study was to investigate the efficacy of the RNAi method for ablation of PLB gene expression and restoration of Ca2+ uptake function in cultured neonatal rat cardiac myocytes in which SERCA2 protein levels were decreased. Myocytes were transfected with 21-nucleotide duplexes of small interfering RNA (siRNA) targeting PLB (30 nmol/l) or with scramble sequence using a haemagglutinating virus of Japan (HVJ) envelope vector. Administration of PLB siRNA resulted in the reduction of PLB mRNA level to approximately 6% of that observed after administration of scramble siRNA group at 12 h after transfection. Further, PLB protein levels in the PLB siRNA groups were 12% of that in cells treated with scramble siRNA on day 2, and the mRNA and protein levels for SERCA2 and calsequestrin were not affected. In addition, Ca2+ uptake affinity was increased in total homogenates from the PLB siRNA group (a 29% decrease in EC50 value when compared with scramble siRNA group). Finally, PLB siRNA restored Ca2+ uptake affinity following hydrogen peroxide-induced decreases in SERCA2 and PLB mRNA expression. These results demonstrate that PLB-targeted RNAi inhibited endogenous PLB expression in neonatal rat myocytes and restored Ca2+ uptake affinity in cardiac myocytes in which SERCA2 protein levels were decreased. This technique may represent a novel therapeutic strategy for heart failure. (C) 2004 Elsevier Ltd. All rights reserved.