AAV‐mediated knockdown of phospholamban leads to improved contractility and calcium handling in cardiomyocytes

Abstract

Reduced contractility due to dysregulation of intracellular calcium (Ca(2+)) is a common pathologic feature of chronic heart failure. Calcium stores in the sarcoplasmic reticulum play a major role in regulating cardiac contractility. Several animal models of heart failure have been treated by altering the regulation of the sarcoplamic reticulum ATPase through ablation or down-regulation of its inhibitor peptide, phospholamban (PLN). We have designed two small hairpin RNAs (shRNAs) to block the synthesis of PLN via RNA interference. These were tested in cell culture using a co-transfection assay and using adeno-associated virus (AAV)-mediated delivery to cardiomyocytes. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blots were used to measure reduction in PLN mRNA and protein levels. Reduction of PLN was also documented by indirect immunofluorescence. Free cytosolic calcium and contractile properties of transduced cardiomyocytes was examined on fura-2-loaded cells. Direct cardiac injection was used to deliver AAV1-shRNAs to mice, and reduction of PLN was measured by indirect immunofluorescence. Both siRNAs led to significant reduction of PLN RNA and protein levels in cultured cells. Down-regulation of PLN led to enhanced cell shortening and relaxation and to a decrease in the time constant of calcium decay, signs of improved contractility and calcium handling. In the hearts of AAV-infected mice, shRNA-transduced cells showed significant reduction in the level of PLN. Our results suggest that AAV-delivered shRNAs mediated physiologically significant suppression of phospholamban that may be useful in combating the effects of chronic heart failure.