Microarray screen identifies miRNAs that are regulated during cardiac hypertrophy. (A) Up- and down-regulated miRNAs in phenylephrine (PE) treated neonatal cardiomyocytes. (B) Fold change of miRNA expression in 2 week thoracic aortic banding (TAB) hearts. (C) Northern blot analysis of miRNAs using equal amounts of total RNA isolated from rat neonatal cardiomyocytes treated with various hypertrophic stimuli. PE, phenylephrine; LIF, leukemia inhibitory factor; FBS, fetal bovine serum. (D) Northern blot analysis of miRNAs using equal amounts of total RNA isolated from control, 2 week, or 4 week TAB mice. tRNAs were used as a loading control. (E) RT-PCR analysis for miR-18b expression in PE or LIF treated cardiomyocytes. U6 expression served as control. (F) Gross heart morphology of sham, 2 and 4 week TAB hearts, and RT-PCR analysis for hypertrophic genes using total RNA isolated from those conditions. GAPDH served as control.

miR-18b and miR-21 regulate cardiomyocyte hypertrophy. (A) Representative images of α-actinin-stained cardiomyocytes transfected with LNA antisense oligonucleotides against indicated miRNAs (upper panels) or miR-duplexes (lower panels). GFP was used as a control. (B) Quantitative analysis of cardiomyocyte cell size. ∼200 cells immunostained with anti-α-actinin antibody from each treatment were randomly chosen for surface area measurement. Results presented as relative cell area compared to controls. (C and D) RT-PCR analysis of hypertrophic marker genes ANF and skeletal muscle α-actin (sk-α-actin) using total RNA isolated from cardiomyocytes treated with indicated LNA oligos, or miRNA duplexes. (E) Western blot analysis of α-actinin expression in cardiomyocytes treated with indicated LNA oligos, miRNA duplexes, or GFP. β-tubulin serves as a loading control. Error bars represent standard deviation; * p < 0.05; ** p < 0.01.