Effects Of AsPLB Gene Transfection Using Ultrasoundmediated Microbubble Destruction

Part One Increase of capillary permeability and enhancement of naked plasmid DNA transfection in myocardium using ultrasound-mediated microbubble destruction in miceBackground:Diagnostic methods and therapeutic means to heart diseases have great improvement in recent years. But it still remains the leading cause of death across all populations. Cardiac gene therapy has already been investigated in experimental and some clinical studies. The success of gene therapy was determined by the effect of therapeutic target genes, efficiency of vector and delivery system. Direct intramyocardium injection, left ventricular cavity injection or intracoronary perfusion was common used. Some of them need surgery to open chest wall, or induce cardiac arrest, or clamp the aorta and pulmonary artery for a brief period of time. They are limited by procedure related risks.Sonoporation was defined as creation of transient, non-lethal holes in the plasma membrane with the assistance of ultrasound. It was reported extracellular molecules are able to diffuse through these holes thus facilitate gene transfer. Intravenous injection of microbubble act as cavitation nuclei enhances sonoporation effect and aid in a wide range of ultrasound-mediated drug delivery applications.Objective:This study was taken to optimize ultrasound parameters of increase of capillary permeability in myocardium by ultrasound-mediated destruction of albumin perfluorocarbon microbubbles via systemic injection of Evans blue dye. We also extended the method for plasmid deoxyribonucleic acid (DNA) transfection.Methods:We constructed albumin perfluorocarbon microbubbles. Evans blue was injected intravenously into mice via tail vein. We evaluated the effects of ultrasound parameters known to influent microbubble destruction, including ultrasound probe, ultrasound frequency and ultrasound mechanical index, on Evans blue extraction. The parameters caused greatest extent of Evans blue extraction was used to perform plasmid DNA transfection. X-gal staining andβ-galactosidase quantification were made to detect gene expression. In addition, mice left ventricular systolic function evidenced by echocardiography and gene expression in liver, lung and kidney were evaluated.Results:1. Optimal ultrasound parameter of this instruction was S3 transduce, 1.3MHz and mechanical index 1.6, with electrocardiogram triggering. Evans blue dye extraction reached utmost extent in this condition combined with microbubbles compared with dye injection without ultrasound and microbubbles (251.59±16.4 vs 30.87±4.26, p<0.05). There was no red blood cells effusion in tissue sections.2. Expression ofβ-galactosidase was found in heart in the optimal parameter 10 days later, and the activity was markedly increased compared with plasmid injection olne or combined with ultrasound olne. Expression ofβ-galactosidase was not found 20 days later. 3. Expression ofβ-galactosidase was not found in liver, lung and brain by X-gal staining, but found in tubular epithelial cells of kidney in each group, including the control group without plasmid injection, indicated endogenousβ-galactosidase activity in kidney .4. Mice left ventricular systolic function was not significantly altered 10 days and 20 days after insonation and injection of plasmid DNA.Conclusion:1. Ultrasound destruction of microbubbles can increase capillary permeability significantly in myocardium under optimal parameter.2. Although capillary permeability increased, mice were secure from normal left ventricular systolic function and no red blood cells effusion.3. Ultrasound mediated-microbubble destruction enhanced reporter gene transfection to myocardium by injection of plasmid-microbubble mixture with simultaneous sonication. Targeted transfection maybe achieved by sonication through chest wall. Part Two Effects of asPLB gene transfection using ultrasound-mediated microbubble destructionBackground:Phospholamban (PLB) is a critical regulator of the cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA_2a) activity and cardiac contractility. Dephosphorylated PLB inhibits SERCA_2a activity, whereas phosphorylated PLB dissociates from SERCA_2a and leads to enhanced contractibility. PLB can be phosphorylated at the serine16 residue by cAMP-dependent protein kinase and at the threonine17 residue via CaMKâ…¡. It has been shown that reduced serine16 phosphorylation of PLB (Serl6-PLB) in the failing rat myocardium is a major contributor to decreased SERCA_2a activity. In vitro and in vivo studies have demonstrated that inhibition of PLB expression, increase the phosphorylated PLB in myocardium can enhance SERCA_2a activity and then restore left ventricular systolic function in failing heart.Transfection of antisense PLB (asPLB) to myocardium of myocardial infarction rats can inhibit up-expression of PLB, and improve the cardiac function. But in vivo transfection of an exotic gene to heart usually requires surgical procedure or catheter-based endomyocardial approaches. They are invasive and expensive. Recently, ultrasound-mediated microbubble destruction has been proposed as a new technique for site-specific gene delivery as a noninvasive approach. Microbubbles act as the cavitation nuclei to focus ultrasound energy by lowering the threshold of sonoporation, which means transient ultrasound-induced increase in permeability of cell membrane or the capillary wall.Objective:We design to estimate the effect of gene transfer of pAAV-antisense phospholamban (pAAV-asPLB), using ultrasound mediated microbubble destruction, on the left ventricular function, PLB and Serl6-PLB protein expression, cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA_2a) protein level and activity in myocardial infarction (MI) mice.Methods:MI mice were generated by ligating the left anterior descending coronary artery. Microbubbles were prepared by sonicated perfluorocarbon gas with dextrose and albumin. pAAV-LacZ was used as a reporter gene to determine the efficiency and localization of transfection. A mixture of pAAV-asPLB plasmid and microbubbles was injected via tail vein while the heart was simultaneously exposed to ultrasound via transthoracic insonation. Mice were divided into 9 groups as follows: normal control, sham-operation, MI+saline, MI+LacZ, MI+LacZ+US, MI+LacZ+MB+US, MI+asPLB, MI+asPLB+US, MI+asPLB+MB+US. Three weeks later, left ventricular ejection fraction (LVEF) and fraction shortening (FS) were measured by echocardiography. PLB, Ser16-PLB, SERCA protein level and SERCA activity were examined.Results:1. Expression ofβ-galactosidase was found in heart in MI+LacZ+MB+US group, not found in liver, lung and brain in each LacZ group, but found in tubular epithelial cells of kidney in each LacZ group and Ml+saline group, indicated endogenousβ-galactosidase activity in kidney .2. Compared with sham-operation, SERCA protein level was not significantly altered in MI mice; but PLB protein level increased significantly, Serl6-PLB decreased significantly; left ventricular diameter enlarged, FS and LVEF markedly depressed in MI groups. SERCA activity was decreased significantly in MI groups except MI+asPLB+MB+US.3. Compared with Ml+saline, PLB, Serl6-PLB, SERCA protein level and SERCA activity were not changed markedly in MI+asPLB, MI+asPLB+US. No significant improvement in left ventricular diameter, FS and LVEF in these two groups.4. LVEF (48.2±5.18% vs 39.1±5.38%, p<0.05), FS (19.6±2.59% vs 16.0±2.29%, p<0.05), SERCA activity (3.00±0.29 vs 2.12±0.30, p<0.05) and Serl6-PLB protein level (0.8±0.25 vs 0.46±0.18, p<0.05) were increased in MI+asPLB+MB+US group while PLB protein level (1.45±0.38 vs 2.05±0.31, p<0.05) was decreased compared with MI+saline. SERCA protein level was not statistical difference.Conclutions:1. Ultrasound alone can not achieve effective gene transfer in myocardium by systemic injection of plasmid.2. Ultrasound-mediated microbubble destruction can enhance exotic gene transfer to myocardium. Targeted transfection maybe acquired by systemic injection of plasmid-microbubble mixture with simultaneous sonication through chest wall.3. asPLB gene transfection can be achieved by ultrasound-mediated microbubble destruction. Effective transfection can partly restore heart function in MI mice, which inhibited myocardial PLB protein expression, restored phosphorylation of PLB on Ser16 site, and restored myocardial SERCA activity. It had no effects on myocardium SERCA protein level.