Thomas E. Callis, Kumar Pandya, Hee Young Seok, Ru-Hang Tang, Mariko Tatsuguchi, Zhan-Peng Huang, Jian-Fu Chen, Zhongliang Deng, Bronwyn Gunn, Janelle Shumate, Monte S. Willis, Craig H. Selzman, Da-Zhi Wang
(A) Transcripts for αMHC, βMHC, and ANF were detected by real-time PCR in 4-month-old hearts from control and miR-208a Tg mice (n = 5 per genotype). Data are the mean fold change in expression ± SEM. *P < 0.01. (B) Left: Western blot analysis of total MHC and βMHC protein levels in adult control and miR-208a Tg hearts. Right: Quantitative analysis of fold change in protein levels. *P < 0.01. (C) Top left: RT-PCR was used to detect αMHC, βMHC, and ANF transcripts in wild-type hearts following 3 weeks thoracic aortic banding (TAB) or in surgical sham hearts, which were used as controls. Bottom left: miR-208a and miR-208b were detected by Northern blot analysis. U6 served as a loading control. Right: Quantitative analysis of fold change in expression of mRNAs and miRNAs. *P < 0.01. (D) Northern blot and quantitative analysis of expression of miRNAs using hearts from control and miR-208a Tg mice. (E–H) Isolated rat neonatal cardiomyocytes were transduced with miR-208a and control adenoviruses (Ad-208 and Ad-Cntl, respectively) or transfected with oligonucleotides antisense to miR-208a or control oligonucleotides (2′Ome-208a and 2′Ome-Cntl, respectively). (E) Cardiomyocytes were stained for α-actinin or βMHC proteins. Original magnification, ×200. (F) Fold change in mean cell area ± SEM of α-actinin–immunostained cardiomyocytes were treated with adenoviruses or oligonucleotides (n = 100 cells/condition; **P < 4 × 10–12). (G) Fold change in mean fluorescent intensity ± SEM of βMHC-immunostained cardiomyocytes treated with adenoviruses or oligonucleotides (n = 100 cells/condition; #P < 3 × 10–7). (H) Cardiomyocytes were treated with adenoviruses or antisense 2′O-methyl oligonucleotides and were scored for ANF staining (n ≈ 425 cells/condition).