238. Knock down Phospholamban To Improve Heart Function by AAV_Mediated shRNA
Authors: Xiaochun Lu, Guang Yang, Xiaoying Li, Xiaobing Wu
Journal: Molecular Therapy (2006)

Background:Congestive heart failure is the syndrome of final stage in most heart diseases, which prognosis is worst. Alterations of calcium_regulatory are the certain access, whereas function and regulation of sarcoplasmic reticulim Ca2+_ATPase are the definitive factors to maintain the calcium equilibrium in cardiomyocyts, and abnormalities of calcium cycling in the failing heart. Phospholamban is a key gene, which control the homoeostasis of Ca2+ by regulating sarcoplasmic reticulum Ca2+ _pump(SERCA2a) in cardiac muscle. Overexpression of an antisense phospholamban construct or a dominant_negative mutant of phospholamban has been shown to improve both systolic and diastolic function. RNA interference (RNAi) is a form of posttranscriptional control in which the double_ stranded RNA (dsRNA) leads to specific degradation of mRNAs with complementary sequence. To assess value in the gene therapy of congestive heart failure, A recombinant Adeno_Associated Virus, which delivery the short 21nt stem hairpin RNA duplexes to knock down phospholamban in cardiomyocyts, was constructed and employed in animal experiment. Methods: We constructed three A AV plasmids, named pSNAVpRi1, pSNAV_pRi2 and pSNAV_pRi3, in which short 21nt stem hairpin RNA duplexes under the control of the U6 snRNA promoter were cloned into pSNAV_1 by PCR or a big adaptor. Transfections of these siRNA plasmids were performed with Lipofectamine into cultured rat cardiac myocytes. Western blot were performed to compare the difference between the expression quantity of phospholamban. Then two recombinant Adeno_Associated Virus, were constructed according to the strategy a recombinant helper virus infecting a carrier cell, rAAV_pRi2 and rAAV_pRi3. Abdominal aortic coarctation rat model that aortery coarctated diameter 0.7mm just above double renal arteris were prepared. After heart failure affirmed, the rAAV were injected into pericardial cavity through diaphragm. The hemodynamics parameters were record by left ventricle ar terial cannulation through carotid artery 10 and 30 days after rAAV injected. Results: three AAV plasmids pSNAV_pRi1, pSNAV_pRi2 and pSNAV_pRi3, could inhibit phospholamban expression about 10%, 14.5% and 23.6%. Then we produced the recombinant adeno_ associated virus rAAV_pRi2 and rAAV_pRi3. The hemodynamics state of rAAV_ pRi2 and rAAV_ pRi3 group was equal to that of control group in 10 days after virus delivery , but better in 30 days. In 30 days after virus delivery, two AAV vector could inhibit phospholamban expression about 74.56% and 91.46%, whereas the SERCA2a mRNA level were no different in control group and two virus delivery groups. In rAAV_ pRi2 group, the the LVSP, +dp/dt and _dp/dt were improved 50.4%, 159.3% and 179.4% than control group.In rAAV_ pRi3 group, the the LVSP, +dp/dt and _dp/dt were improved 80.5%, 223% and 278.4%, meanwhile the LVEDP lower 84.7% than control group. Conclusion: Those two recombinant adeno_associated virus, rAAV_ pRi2 and rAAV_ pRi3 effectively inhibit phospholamban expression. Those products injected aortic coarctation rat can improve the hemodynamics state of heart failure rats.