Development of novel heart failure therapy by using gene transfer into cardiac myocytes

1)将SERCA 2基因导入心肌细胞以开发新的心力衰竭治疗方法我们利用腺相关病毒和慢病毒建立了携带人SERCA 2 cDNA的基因转移系统。通过将这些构建体感染到心肌细胞中，我们观察到SERCA 2 mRNA增加了3倍，SERCA 2蛋白增加了2倍。在转染的心肌细胞中，Ca 2+摄取能力比用不携带SERCA 2 cDNA的对照载体感染的组增强40%。2）RNA干扰技术抑制受磷蛋白表达治疗心力衰竭受磷蛋白是SERCA 2蛋白Ca ~（2+）摄取功能的关键调节蛋白。受磷蛋白的抑制增加了Ca 2+摄取功能。本研究建立了受磷蛋白特异性RNA干扰方法。利用HVJ包膜将受磷蛋白基因编码区特异性的双链21核苷酸序列导入新生大鼠心肌细胞。受磷蛋白siRNA的作用对靶mRNA具有高度基因特异性，并将其mRNA水平降低至对照组的10%。受磷蛋白的含量也显著下降至对照组的10%。重要的是，Ca 2+吸收动力学发生了变化，使效率提高了38%。受磷蛋白RNAi的这些有益作用也在过氧化氢诱导的心力衰竭模型中得到证实。受磷蛋白消融组的钙摄取恢复，我们的数据表明，钙转运蛋白的遗传调节在心力衰竭的治疗中具有治疗益处。

1) Development of novel heart failure therapy by introducing SERCA2 gene into cardiac myocytesWe have developed gene transfer system bearing human SERCA2 cDNA using adeno-associated virus and lenti-virus. By infecting these constructs into cardiac myocytes, we observed 3-fold increase of SERCA2 mRNA and 2-fold of SERCA2 protein. In the transfected myocytes, Ca2+ uptake capacity was enhanced by 40% in compared with the group infected with control vector that did not harbor SERCA2 cDNA. We are now under investigation of the effect of the gene transfer on the in vivo cardiac function in the rat.2) Development of heart failure therapy by inhibiting the phospholamban expression using a RNA interference methodPhospholamban is a key regulatory protein for Ca2+ uptake function of SERCA2 protein. Inibition of phospholamban increases the Ca2+ uptake function. In this research project, we have developed phospholamban-specific RNA interference method. Double strand 21 ribonucleotide sequence specific for coding region of phospholamban gene was introduced into rat neonatal cardiac myocytes using HVJ envelope. The effect of phospholamban siRNA was highly gene specific for target mRNA and reduced its mRNA level to 10% of control group. The anount of phospholamban protein was also significantly decresed to 10% of control. Importantly, Ca2+ uptake kinetics was shifted to increase the efficiency by 38%. These beneficial effect of phospholamban RNAi was also demonstrated in the hydrogen peroxide-induced failing heart model. Decreased Ca2+ uptake was restored in the phospholamban ablation group.Our data suggest that genetic modulation of Ca2+ transporting protein has a therapeutic benefit in the treatment of heart failure.