Partial Restoration of Left Ventricular Systolic Function by asPLB Gene Transfer Using Ultrasound-Mediated Microbubble Destruction

Abstract 

In vitro and in vivo studies have demonstrated that inhibition of phospholamban (PLB) expression in myocardium can restore left ventricular systolic function in failing heart. Ultrasound mediated microbubble destruction provides a new option for noninvasive gene transfer in heart. In this study, we transfered pAAV-antisense phospholamban (pAAV-asPLB) to the hearts of myocardial infarction (MI) mice, using ultrasound mediated microbubble destruction. Then we estimated the protein levels of PLB, Ser16-PLB and cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA). The left ventricular ejection fraction (LVEF), fraction shortening (FS) and SERCA activity were measured as well. MI mice were generated by ligating the left anterior descending coronary artery. Microbubbles were prepared by sonicated perfluorocarbon gas with dextrose and albumin. A mixture of pAAV-asPLB plasmid and microbubble was injected via tail vein while the heart was simultaneously exposed to ultrasound via transthoracic insonation. Three weeks later, 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 Ser16-PLB protein level (0.8±0.25 vs 0.46±0.18, p<0.05) were increased while PLB protein level (1.45±0.38 vs 2.05±0.31, p<0.05) was decreased compared with the MI mice with saline injection. The above parameters in MI mice with only pAAV-asPLB plasmid injection or pAAV-asPLB plasmid combined with ultrasound alone were not significantly improved. pAAV-LacZ was used as a reporter gene to determine the efficiency and localization of transfection. The expression of β-galactosidase was not found in liver, lung and brain, but found only in tubular epithelial cells of kidney and found in heart. These results confirm that asPLB gene transfection can be achieved by ultrasound mediated microbubble destruction with organ specificity. The effective transfection can partly restore heart function in MI mice. (E-mail: s0hu0001@hotmail.com)

Key Words: Antisense phospholamban, Cardiac sarcoplasmic reticulum Ca²⁺ ATPase, Ultrasound mediated microbubble destruction, Left ventricular function, Myocardial infarction