Myocardin is sufficient for a smooth muscle-like contractile phenotype

Abstract

Background: Myocardin (Myocd) is a strong coactivator that binds the serum response factor (SRF) transcription factor over CArG elements embedded within smooth muscle cell (SMC) and cardiac muscle cyto-contractile genes. Here, we sought to ascertain whether Myocd-mediated gene expression confers a structural and physiological cardiac or SMC phenotype.

Methods and results: Adenoviral-mediated expression of Myocd in the BC(3)H1 cell line induces cardiac and SMC genes while suppressing both skeletal muscle markers and cell growth. Immunofluorescence microscopy shows that SRF and a SMC-like cyto-contractile apparatus are elevated with Myocd overexpression. A short hairpin RNA to Srf impairs BC(3)H1 cyto-architecture; however, cotransduction with Myocd results in complete restoration of the cyto-architecture. Electron microscopic studies demonstrate a SMC ultrastructural phenotype with no evidence for cardiac sarcomerogenesis. Biochemical and time-lapsed videomicroscopy assays reveal clear evidence for Myocd-induced SMC-like contraction.

Conclusions: Myocd is sufficient for the establishment of a SMC-like contractile phenotype.

Figure 1. Myocd inhibits proliferation of BC₃H1 cells

A. Phase contrast micrographs of control- and Myocd-transduced cells 5 days post-transduction. B. BC₃H1 cell growth over 5 days following control (open bars) or Myocd (closed bars) adenoviral transduction. Bars here and below indicate standard error of the mean. Inset verifies daily Myocd mRNA expression. C. Cyclin D1 protein expression in BC₃H1 cells ± Myocd. D. Luciferase activity (ratio of luciferase to renilla) in BC₃H1 cells ± Myocd using a cyclin D1 luciferase reporter in low or high serum.

Figure 2. Modulated muscle marker profile in growing versus differentiated BC₃H1 cells

A. RT-PCR analysis of endogenous mRNAs in growing versus differentiated (sarcomeric) BC₃H1 cells. B. Northern blot of SRF in growing vs differentiated BC₃H1 cells. Hprt is a house-keeping gene. C. Immunostaining of human CNN1 (in red) in growing (left) vs differentiated (right) BC₃H1 cells carrying a BAC with human CNN1. Nuclei are stained blue with DAPI. Final magnifications are 400x.

Figure 3. Myocd regulated muscle markers in BC₃H1 cells

A. BC₃H1 cell mRNA expression of smooth muscle cell (SMC), cardiac muscle cell (CaMC), and skeletal muscle cell (SkMC) markers ± 100 moi Myocd. B. Muscle marker proteins ± 100 moi Myocd. Alpha tubulin (TUBA) serves as an internal loading control.

Figure 4. Myocd induces structural attributes of differentiated SMC

BC₃H1 cells transduced (100 moi) with control (a), shSRF (b), Myocd (c) or Myocd + shSRF (d) adenovirus for 72 hr and stained for SRF (green) or cyto-contractile fibers using phalloidin (red). All photomicrographs were taken at same exposure time.

Figure 5. Myocd induces SMC-like myofilaments in BC₃H1 cells

A. Ultrastructure of in vivo mouse aortic SMC showing myofilament array (white arrow). B. BC₃H1 cells transduced with control adenovirus. C. BC₃H1 cells transduced with Myocd. Note bundle of smooth myofilaments (black arrow). Magnifications, 15,000x . D. Quantitative measure of smooth myofilaments. Data represent semi-quantitative scoring (see Supplemental Methods at http://atvb.ahajournals.com) of more than 80 cells from two independent experiments.

Figure 6. Myocd-induces SMC-like contraction

A. Expression of SM proteins in BC₃H1 ± increasing amounts of Myocd adenovirus. B. Expression of pMLC₂₀ in BC₃H1 ± Myocd. C. Control-transduced BC₃H1 cells (a, b) and Myocd-transduced cells (c, d) before (a, c) and after (b, d) 8 minute stimulation with 75 mM KCl. Arrows point to cells exhibiting changes in cell size. D. Quantitative measureof contractility in contro l- vs Myocd-transduced BC₃H1 cells.