Fig. 2.
Myocardin-deficient aortic SMCs exhibit markedly diminished expression of SMC contractile proteins and induction of ECM. (A–J) Four months following tamoxifen treatment, aortic sections prepared from MyocdF/F control mice (A–E) demonstrate robust expression of SMA (green), SMMHC (red), SM22α (red), calponin-h1 (red), and MLCK (green). By contrast, SMC contractile proteins are barely detectable in aortic sections prepared from tamoxifen-treated SMMHC-CreERT2/MyocdF/F mutant mice (F–J). (K) qRT-PCR demonstrating 80–90% decrease in expression of genes encoding myocardin (Myocd), SMMHC, SM22α, SMA, caldesmon, and calponin-h1 in tamoxifen-treated SMMHC-CreERT2/MyocdF/F mutant mice (red bars) compared with MyocdF/F control mice (black bars). Data are expressed as mean gene expression (arbitrary units) ± SEM. (L) Representative ethidium bromide-stained agarose gel showing relative levels of gene expression of myocardin, SRF, and SMC contractile genes (SMA, SMMHC, SM22α, Caldesmon) in qRT-PCR amplified mRNA harvested from tamoxifen-treated MyocdF/F (Control) and SMMHC-CreERT2/MyocdF/F mutant (Mut) mice. (M–T) Aortic sections harvested from tamoxifen-treated MyocdF/F control (M–P) and SMMHC-CreERT2/MyocdF/F mutant (Q–T) mice were stained with Masson’s trichrome (M and Q) or immunostained with antibodies that detect tropoelastin (N and R), fibronectin (O and S), or laminen (P and T). Masson’s trichrome staining reveals loss of SMC cytoplasm (red stain) and induction of medial fibrosis (blue stain) in the Myocd mutant aorta (Q) compared with the control (M). Tropoelastin immunostaining (black) reveals preservation of lamellar architecture (N and R). However, a dramatic induction of fibronectin (red) and laminin (red) are observed in the aorta of tamoxifen-treated Myocd mutant mice (S and T) compared with controls (O and P). (U–V) Electron micrographs of sections prepared from the ascending aorta harvested 4 mo following tamoxifen treatment of MyocdF/F mice (U and V) and SMMHC-CreERT2/MyocdF/F mutant mice (W and X). Abundant myofibers (Myo) are observed throughout the cytoplasm of the control mouse (U and V). By contrast, contractile fibers are not observed in the cytoplasm of vascular SMCs of SMMHC-CreERT2/MyocdF/F mutant mice (W and X). However, abundant fibrinous collagen (Col) is observed in the extracellular spaces (W and X). U, V, W, and X, original magnification, 50,000×.