Molecular changes induced by reduced miR-1/133a abundance.

<p>mRNA as well as protein expression of several previously described direct miR-1 or miR-133a target molecules was analyzed in heart tissue of WT and respective single miR-1/133a cluster knock-out animals to validate the regulation of these targets in our in vivo models. In the heart of single cluster knock-out animals the miR-1 target myocardin was not upregulated at protein level (A) or mRNA level (C). This applied to the embryonic heart (eH) specific isoform of myocardin as well as to the other isoforms exclusively detected in the adult heart. In contrast the miR-133a target SRF is significantly upregulated in the heart of miR-1-1/133a-2 knock-out mice (B, D). In line with up-regulation of SRF, we also confirmed the Affymetrix-analysis based finding that smooth muscle marker genes were upregulated in the adult heart of miR-1-1/133-2 mice (E). We detected significant upregulation of the miR-1 target Kcne1 in both single cluster knock-out (F). The miR-1 target connexin43 was significantly upregulated in only miR-1-1/133a-2 mutant hearts, corresponding to the more severe loss of miR-1 in this model (G). We did not find upregulation of the previously described miR-1 target IRX5 (H, J). qRT-PCR analysis confirmed the unbiased transcriptome analysis based finding that the proposed Irx5 transcriptional target Kncd2 is not significantly regulated in mutant hearts (K). We observed a downregulation of the previously described miR-1 target gene CAV1.2 (I, L).