Development of novel therapeutic strategy for heart failure by improving Ca2+ transport function of sarcoplasmic reticulum

1)激活SERCA2基因转录的新药物我们筛选了超过80,000种化合物，并在H9C2心肌细胞中挑选了9种激活SERCA2基因转录的化合物。这些化合物应用于主动脉带引起的心力衰竭大鼠，并检测了这些化合物对SERCA2 mRNA水平激活的影响。其中一种化合物将SERCA2 mRNA水平提高了20%。然而，由于其在体内改善心力衰竭的效果还不够高，我们正在进一步搜索化合物库，寻找更有效的化合物。2)SERCA2基因治疗我们开发了携带人类SERCA2 cDNA的慢病毒基因转移系统。我们将该基因构建体导入心肌梗死致心力衰竭大鼠冠状动脉，观察SERCA2基因导入对心力衰竭心功能的影响。基因转染7天后，转染serca2的心肌缩短率从16.5%显著提高到26%，而转染GFP（一种无功能的心脏收缩蛋白）并没有显著提高心肌缩短率（从16%提高到19%）。这些变化与SERCA2 mRNA和蛋白水平的上调相对应，这表明慢病毒SERCA2基因转移系统作为心力衰竭治疗方式的可行性。3)磷蛋白消融方法的建立利用HVJ包膜将磷蛋白基因编码区特异的双链21核糖核苷酸序列导入大鼠新生心肌细胞。磷蛋白siRNA对靶mRNA具有高度的基因特异性，使其mRNA水平降低至对照组的10%。重要的是，Ca2+摄取动力学被转移，使效率提高了38%。在过氧化氢诱导的心力衰竭模型中也证实了磷蛋白RNAi的这些有益作用。磷蛋白消融组恢复Ca2+摄取减少。我们的研究表明，Ca2+转运蛋白的遗传调节在心力衰竭的治疗中具有治疗益处。少

1)New pharmacological agent which activates the transcription of the SERCA2 geneWe have screened over 80,000 chemical compounds and picked up 9 compounds that activate the transcription of the SERCA2 gene in H9C2 cardiac cells. These compounds are applied to rats under heart failure induced by aortic banding and the effect of these compounds on the activation of the SERCA2 mRNA levels examined. One of these compounds increased the SERCA2 mRNA level by 20%. However, because the effect is not sufficiently high to improve heart failure in vivo, we are now further searching the compound library to find out more potent one.2)SERCA2 gene therapyWe have developed lentiviral gene transfer system bearing human SERCA2 cDNA. We introduced the gene construct into rat coronary artery of failing heart induced by myocardial infarction and examined the effect of SERCA2 gene introduction on the cardiac function of failing heart. Seven days after gene transfer, SERCA2-transfected demonstrated the signif … More icant increase of the fractional shortening from 16.5% to 26%, whereas the GFP, a non-functioning protein for contraction of heart, gene transfer did not significantly improve the fractional shortening (from 16 to 19%). These changed were corresponded with the upregulation of the SERCA2 mRNA and protein levels, which suggests the feasibility of the lentiviral SERCA2 gene transfer system as a therapeutic modality of heart failure.3)Development of phospholamban ablation methodDouble 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. 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 research suggests that genetic modulation of Ca2+ transporting protein has a therapeutic benefit in the treatment of heart failure. Less