A new class of engineered regulatory RNAs, amiRs, appears to be safer than shRNAs. For this reason we have used and characterized amiR155-PLBr for silencing of PLB in CM. In accordance with previous reports comparing shRNAs versus amiRs approaches, we found that amiR155-PLBr was expressed at a lower level than shPLBr. The lower activity of the heart-specific CMV-MLC0.26 promoter driving the expression of miR155-PLBr compared to the highly active U6 promoter used for shPLBr delivery is the most reasonable explanation for the observed differences between the two expression systems. We cannot rule out, however, that other factors, for example individual structural characteristics, cause the differential expression of amiR155-PLBr and shPLBr. The lower expression of amiR155-PLBr was linked to distinctly less pronounced PLB silencing. Interestingly, when both amiR155-PLBr and shPLBr were expressed at similar levels, achieved by about 10-fold increase of scAAV6-amiR155-PLBr vector dose, PLB silencing was similar. Thus, our data suggest,AZD4635 that amiR155-PLBr and shPLBr have per se similar silencing efficiency. This, however, is somewhat contradictory to previous reports. McBride et al. and Mazcuga et al. found comparable silencing efficiency of amiRs and shRNAs despite the lower expression they found for amiRs. Other studies confirmed these data and reported higher efficacy of amiRs as compared to shRNAs. So far, the mechanisms underlying the differing activities of amiRs and shRNA are not well understood. Differences in stability and processing to generate mature siRNA molecules may be important factors. In particular, the specific structure of amiRs mimicking the naturally occurring cellular miR structure has been assumed to be a determinant that makes amiRs more efficient than shRNAs. In addition, the use of different termination signals and the distance between the promoter, the shRNA/amiR sequence and the termination signal may affect the respective silencing efficiency. Although no in-depth analysis has been carried out yet, sequence-specific determinants may play an important role as well. This would explain why, in some cases, amiRs are more efficient than shRNAs, while in other cases, amiRs and shRNAs have similar KR-33493 activity or amiRs may even be less efficient. We confirmed that the silencing efficiency of amiR155-PLBr used here was comparable to that using a miR-30 scaffold. Hence, the miR-155 scaffold does not affect the inhibitory activity of amiRs. The repression of the PLB protein in CM by amiR155-PLBr by 50% and 70% at days 7 and 14 post transduction, respectively, was considerably lower than that induced by shPLBr. Nevertheless, the amiR155PLBr-mediated PLB silencing was functionally as efficient as that of shPLBr, i.e. the increase of SERCA2a-mediated SR Ca2+ transport was similar for both small regulatory RNAs. Hence, the diminished PLB silencing by amiR155-PLBr appears to be functionally irrelevant in the case studied here. The effect observed here may be explained by a functional threshold for PLB-induced inhibition of SERCA2a. Both silencing strategies diminished PLB to a level below this value. Thus, we have carried out a proteomic analysis and validated some of our findings by semiquantitative Western blot analysis.