Construction Of Phospholamban Antisense RNA Recombinant Adeno-associated Virus Vector And Its Effect On Heart Failure In STZ-induced Diabetic Rats

Part IConstruction of phospholamban antisense RNA recombinant adeno-associated virus vector and its effect in rat cardiomyocytesObjectiveTo construct a recombinant adeno-associated virus vector containing gene encoding phospholamban antisense RNA (rAAV-asPLB), as well as analyse its effect on expression of PLB, activity of sarco-endoplasmic reticulum(SR) Ca²⁺-ATPase, and the change of intracellular free Ca²⁺ concentration ( [Ca²⁺]i ) in rat cardiomyocytes. Methods 1. Construction of recombinant adeno-associated virus: Rat PLB cDNA fragment(695 bp ) was synthesized by reverse transcription-polymerase chain reaction ( RT-PCR) from total RNA isolated from the heart of Wistar rats. The synthesized PLB cDNA was subsequently digested by BamH I and EcoR I simultaneously, while the adeno-associated virus plasmid pAAV-MCS digested by corresponding restricted endonuclease enzyme. The pAAV-asPLB was generated by cloning the PLB cDNA into EcoR I / BamH I sites of pAAV-MCS reversed orientation relative to the promoter. Identification of the gene was confirmed by restriction enzymes digestion and sequencing. The rAAV-asPLB was constructed by plasmid pAAV-asPLB and pAAV-RC and pHelper co-transfected into AAV293 cell. At the same time, A viral production positive control (rAAV-LacZ) and negative control were performed.2. Rat cardiomyocytes culture. Rat nenonatal cardiomyocytes were isolated, cultured and infected with the rAAV at a multiplicity of infection ( MOI ) of 100 for 48 h..Cardiomyocytes were divided into 3 groups: â‘ normal control; â‘¡infected with rAAV- LacZ; â‘¢infected with rAAV-asPLB3. Cell viability was measured by MTT. In Situ β-Galactosidase Staining were performed to observe the transfer efficiency. RT-PCR and Western blot were used to determine the mRNA and protein. The activity of SR Ca²⁺-ATPase, and the change of [Ca ²⁺]i were detected.Results1.The result of pAAV-asPLB restriction enzymes digestion and sequencingdemonstrated that the sequence and direction of PLB cDNA fragment ( 695 bp ) wereprecise. The rAAV-asPLB and rAAV-LacZ were harvested in rAAV-asPLB group andrAAV-LacZ group, respectively.2.Cell viability was no change in rAAV infected groups and normal group.3.The PLB mRNA and protein expression are reduced in rat cardiomyocytes whichtransfected by rAAV-asPLB compared with controls. The activity of Ca²⁺-ATPasewas increased. In rest state, the level of [Ca²⁺]i in rAAV-asPLB transfected groupwas decreased. The level of [Ca²⁺]i was increased when induced by isoproterenol. Conclusion1. The rAAV-asPLB vectors are constructed successfully with AAV Helper-Free System.2. The rAAV vectors were able to transfected into rat cardiolmyocytes effectively and safty.3. rAAV-asPLB vector disrupts the expression of PLB, enhances the activity of Ca²⁺-ATPase, reduced the resting [Ca²⁺]i and enhance the isoproterenol- induced [Ca²⁺]i.ObjectiveDiabetes mellitus ( DM ) was induced by intraperitoneal injection of streptozotocin (STZ) and administered intramyocardiol injection of rAAV-asPLB. The effects of rAAV-asPLB on SR Ca²⁺-ATPase activity and cardiac function in diabetic cardiomyopathy were investigated. MethodsrAAV-asPLB was constructed with AAV Helper-Free System. The DM in male rats were induced by intraperitoneal injection of STZ. Six weeks later, the DM rats were administer intramyocardiol injection of rAAV-asPLB (rAAV-asPLB) or saline (saline) or no treatment (DM). The levels of PLB mRNA and PLB protein, the activity of SR Ca²⁺-ATPase and the hemodynamics parameters were measured 6 weeks later after injection.The normal rats served as control groups. Results1. Left ventricular function was assessed by left ventricle pressure (LVSP), maximal rates of pressure development and decline(±dp/dt_max) and LV end-diastole pressure (LVEDP). Left ventricular dysfunction developed in all the DM groups. In resting state, the depression of LVSP, ±dp/dt_max with an elevation of LVEDP were detected in all the DM rats compared with th...