Myocardin is a primary target of miR-1 in the embryonic heart.

<p>(A, A′) Western blot analysis of increased myocardin expression in <i>miR-1/133a</i> dKO embryonic hearts at E10.5 compared to WT. (B, B′) No increase of SRF protein expression in <i>miR-1/133a</i> dKO embryonic hearts at E10.5 compared to WT. (C, D) Putative miR-1 (C) and mir-133a (D) WT and mutant binding sites located in the 3′ UTRs of myocardin and Kcnmb1 mRNAs were cloned into luciferase reporter vectors. (E, F) miR-1 (E) and miR-133a (F) mediated suppression of luciferase activity via WT but not mutant miRNA binding sites located in myocardin (E) and (F) Kcnmb1 mRNAs. Embryonic cardiomyocytes were isolated from embryonic hearts (E11.5-13.5) (G, H, H′). Transfection of miR-1 or of scrambled control (scr) into embryonic cardiomyocytes confirms miR-1 mediated repression of endogenous myocardin transcripts (qRT-PCR; G) and of myocardin protein (Western Blot; H, H′). (I, J, J′) Transfection of miR-133a into embryonic cardiomyocytes confirms miR-133a mediated repression of Kcnmb1 mRNA (qRT-PCR; I) and Kcnmb1 protein (Western Blot; J, J′).