Avoiding undesirable side effects is critical for therapeutic applications since they restrict application of the therapeutic agent in humans. Importantly, shPLBr, but not amiR155-PLBr, induced the expression of the IFN-regulated proteins PrP and Gal3BP and the proinflammatory genes STAT1 and STAT3, accompanied by STAT1 activation via phosphorylation at the Tyr701 residue. Both STAT proteins are part of the Janus kinase /STAT pathway, which mediates transduction of stress signals from the plasma membrane to the nucleus. Hence, our results demonstrate that long-term and high level shPLBr expression can induce undesirable induction of stress response genes in CM, probably via IFN-dependent pathways. This activation may be avoided by the use of amiR155-PLB. We also found dysregulation of several other proteins after shPLBr and amiR155-PLBr vector treatment. In most cases this may be SHP099 hydrochloride caused by adaptation of the cellular protein network to the changed cellular metabolism as a result of PLB downregulation. Whether these findings will have relevance for the in vivo application of the vectors is currently not clear. It should, however, be considered in follow up studies. We also detected alterations in the expression of cardiac miRs after transduction of CM with the shRNA and amiR155 expressing AAV vectors used in this study. Compared to untreated CM, the miRs levels were distinctly reduced, indicating that RNAi vectors have affected the miR biosynthesis. Importantly, miR downregulation occurred through treatment with PLB-silencing as well as its respective shRNA and amiR155 expressing control vectors. Hence, the suppressing effect was not restricted to shPLBr and amiR155-PLBr but represented a common outcome of AAV vector-mediated transduction and expression of interfering RNAs. High shRNA expression can interfere with the cellular miR expression and thus high vector doses used here, leading to strong shRNAs and amiR155 expression, may be the reason for our observations. Vector transduction effects, however, have also been taken into account. In a recent study, Bish et al. detected dysregulation of several cardiac miR after AAV6-shPLB application to dogs. In contrast to our observations, SSR240612 they detected downand up-regulation of miRs. The state of cellular differentiation, speciesspecific differences as well as differences between the in vitro and in vivo situation may be possible explanations for these differences. In this regard it should also be noted that very strong downregulation of PLB as triggered by RNAi-inducing vectors may be rather undesirable for treatment of heart failure in humans, because in humans PLB is essential for cardiac health and its absence results in lethal heart failure. Thus it is obvious that further improvement of the safety of the RNAi vectors is desirable. An increase of safety might be accomplished by the use of gene expression systems subject to pharmacological regulation. For example, the tetracycline-dependent gene expression system that has already been used successfully for the expression of amiR may be easily adaptable for expression of amiR155-PLBr. The system allows adjustment amiR expression within a therapeutic window, as well as outright on-off switching. Another interesting finding of the miR analysis was a significantly higher expression of miR-21 in CM after treatment with PLB silencing vectors as compared to the respective control vectors.