Figure 2. miR-208a regulates Myh7b / miR-499.

A) Northern blot analysis of heart and skeletal muscle tissue of miR-208a^−/− animals shows that miR-499 expression is specifically extinguished in the heart while the expression in soleus remains unaffected. Compare with lower panel of Figure 1B.

B) Northern blot analysis of heart tissue shows that miR-208a regulates the expression of myh7b/miR-499 in a stoichiometric manner. In miR-208a^+/− animals, miR-499 and myh7b expression is reduced by 50%, while miR-499 and myh7b expression are eliminated in miR-208a^−/− animals. Myh7b and GAPDH were detected by RT-PCR. GAPDH serves as a loading control.

C) Northern blot analysis on cardiac samples of wild-type and miR-208a^−/− (KO) neonate and adult mice. miR-208b and miR-499 are expressed normally in neonatal heart of miR-208a KO mice, whereas neither miRNA is expressed in adult miR-208a KO heart.

D) Mice of the indicated miR-208a genotype were treated with PTU, as indicated, and miRNA expression was detected by Northern blot. PTU treatment increases miR-208b in wild-type animals, and this effect is significantly blunted in miR-208a^−/− animals.

E) Expression of miR-499 in hearts from wild type and MCK-miR-499 transgenic mice detected by Northern blot.

F) Re-introducing miR-499 with an MCK-miR-499 transgene in the miR-208a^−/− background restores expression of miR-208b and β-MHC in response to PTU treatment. Upper panels show Northern blots of hearts from duplicate animals under each condition. Lower panels show β-MHC expression as detected by real time RT-PCR.

G) Fast skeletal muscle troponins (TnnT3 and TnnI2) are up-regulated in hearts of miR-208a^−/− animals. Introduction of the MCK-miR-499 transgene into miR-208a^−/− mice represses troponin expression as in wild-type. Results represent the average values obtained from two animals as detected by real time PCR.