MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice
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
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
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Research Article Cardiology

MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice

Abstract

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have gained status as important regulators of gene expression. Here, we investigated the function and molecular mechanisms of the miR-208 family of miRNAs in adult mouse heart physiology. We found that miR-208a, which is encoded within an intron of α-cardiac muscle myosin heavy chain gene (Myh6), was actually a member of a miRNA family that also included miR-208b, which was determined to be encoded within an intron of β-cardiac muscle myosin heavy chain gene (Myh7). These miRNAs were differentially expressed in the mouse heart, paralleling the expression of their host genes. Transgenic overexpression of miR-208a in the heart was sufficient to induce hypertrophic growth in mice, which resulted in pronounced repression of the miR-208 regulatory targets thyroid hormone–associated protein 1 and myostatin, 2 negative regulators of muscle growth and hypertrophy. Studies of the miR-208a Tg mice indicated that miR-208a expression was sufficient to induce arrhythmias. Furthermore, analysis of mice lacking miR-208a indicated that miR-208a was required for proper cardiac conduction and expression of the cardiac transcription factors homeodomain-only protein and GATA4 and the gap junction protein connexin 40. Together, our studies uncover what we believe are novel miRNA-dependent mechanisms that modulate cardiac hypertrophy and electrical conduction.

Authors

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

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Figure 6

miR-208a and miR-208b repress the expression of Thrap1 and myostatin.

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miR-208a and miR-208b repress the expression of Thrap1 and myostatin. (A...
(A) Sequence alignment between miR-208a and candidate binding sites in the 3′ UTR of Thrap1 and myostatin. (B) Northern blot analysis demonstrated that miR-208a, miR-208b, and miR-124 expression plasmids produced mature miRNAs when transfected into 293T cells. Total RNA from mouse brain and neonatal and adult hearts was included as a control. U6 served as a loading control. (C) 293T cells were transfected with a luciferase reporter designed to detect miR-208a expression (208a sensor), along with the indicated miRNA expression plasmids. A Thrap1 3′ UTR (luc-Thrap1) and a mutated Thrap1 3′ UTR (luc-Thrap1 mutant) were also tested. Values are mean luciferase activity ± SD relative to the luciferase activity of reporters cotransfected with an empty expression plasmid. The dotted line indicates the basal level of luciferase activity in control (i.e., the luciferase vector alone). (D) A luciferase reporter with duplicated Thrap1 binding sites (luc-Thrap1 4×) was cotransfected with miRNA expression plasmids and luciferase activity determined. (E) A luciferase reporter with 4 repeats of the putative myostatin binding site was cotransfected with miRNA expression plasmids and luciferase activity determined. (F) Western blot analysis for Thrap1 and myostatin protein levels in hearts from 4-month-old miR-208a Tg versus control animals and Mir208a–/– versus wild-type animals. GAPDH served as a loading control. *P < 0.05.