Myocardin regulates expression of contractile genes in smooth muscle cells and is required for closure of the ductus arteriosus in mice

Abstract

Myocardin (Myocd) is a potent transcriptional coactivator that has been implicated in cardiovascular development and adaptation of the cardiovascular system to hemodynamic stress. To determine the function of myocardin in the developing cardiovascular system, Myocd(F/F)/Wnt1-Cre(+) and Myocd(F/F)/Pax3-Cre(+) mice were generated in which the myocardin gene was selectively ablated in neural crest-derived SMCs populating the cardiac outflow tract and great arteries. Both Myocd(F/F)/Wnt1-Cre(+) and Myocd(F/F)/Pax3-Cre(+) mutant mice survived to birth, but died prior to postnatal day 3 from patent ductus arteriosus (PDA). Neural crest-derived SMCs populating the ductus arteriosus (DA) and great arteries exhibited a cell autonomous block in expression of myocardin-regulated genes encoding SMC-restricted contractile proteins. Moreover, Myocd-deficient vascular SMCs populating the DA exhibited ultrastructural features generally associated with the SMC synthetic, rather than contractile, phenotype. Consistent with these findings, ablation of the Myocd gene in primary aortic SMCs harvested from Myocd conditional mutant mice caused a dramatic decrease in SMC contractile protein expression. Taken together, these data demonstrate that myocardin regulates expression of genes required for the contractile phenotype in neural crest-derived SMCs and provide new insights into the molecular and genetic programs that may underlie PDA.

Figure 1. Conditional targeting of the Myocd gene.

(A) Schematic representation of the Myocd gene showing exons 7–10 (rectangles). The basic and glutamine-rich domains are encoded by exons 8 and 9. EcoR1 sites (R1) are shown (top panel). The conditional gene-targeting construct contains the PGK-neomycin-resistance (neo) and HSV-thymidine kinase (tk) cassettes. LoxP sites (triangles) flank exon 8 (second panel). The conditionally targeted allele is depicted in the third panel. Targeted allele following Cre-mediated deletion. The position of the DNA probe used for Southern blot analyses is indicated below (black rectangle) (bottom panel). (B) Southern blot analysis of the targeted ES cells demonstrating the WT (11.4-kb) and flanked-by-loxP (Flox) (9.4-kb) alleles. Genotyping of WT (+/+), heterozygous (+/F), and homozygous (F/F) mice. (C) PCR genotyping of control floxed (F/F), control heterozygotes Wnt-Cre transgenic (Cre+/+/F), control heterozygote (+/F), and conditional mutant Wnt1-Cre transgenic (Cre+/F/F) mice. PCR was performed with primers PCR-A and PCR-B (see Methods) to identify products corresponding to WT (1,010-bp) and targeted (1,050-bp) alleles. Primer Cre800F and Cre800R were used to identify 800-bp Cre PCR product. (D and E) Myocardin gene expression in neural crest–derived arteries of Myocd^F/F/Wnt1-Cre⁺ mutant mice. Real time RT-PCR was performed as described in Methods with mRNA harvested from the proximal aorta and carotid arteries of 4 P2 Myocd^F/F/Wnt1-Cre⁺ mice (gray bars) and 4 control littermates (black bars). (D) Data are expressed as mean gene expression (arbitrary units) ± SEM. (E) Representative DNA gel demonstrating GAPDH (291-bp) and myocardin (108-bp) amplified products in control (Cre^–/F/F) and mutant (Cre⁺/F/F) mice.

Figure 2. Wnt1-Cre mediated recombination in neural crest–derived SMCs.

(A and B) Wnt1-Cre transgenic mice were interbred with R26R mice to define pattern of Cre-mediated gene excision. (A) P2 R26R⁺ control mouse demonstrating normal patterning of the cardiac outflow tract and great arteries, pulmonary artery (PA), DA, ascending aorta (AAo), descending aorta (DAo), carotid arteries (CA), and subclavian artery (SC). Original magnification, ×10. (B) P2 Wnt1-Cre⁺/R26R⁺mouse demonstrating β-galactosidase expression (blue stain) in arteries populated by neural crest–derived SMCs. Original magnification, ×10. (C) Transverse section of P2 Wnt1-Cre⁺/R26R⁺ mouse demonstrating the AAo. Most but not all SMCs populating the AAo stain blue. Original magnification, ×200. (D) Transverse section of P2 Wnt1-Cre⁺/R26R⁺ mouse demonstrating robust Wnt1-Cre–mediated recombination in the DA. Original magnification, ×100. (E and F) Frontal sections of an E11.5 Wnt1-Cre⁺/R26R⁺ embryo demonstrating β-galactosidase activity restricted to the endocardial cushions in the right ventricle (RV) (F) and the AAo and DA (E). Original magnification, ×100. (G–J) Immunohistochemical analyses performed with anti-myocardin antibody demonstrating markedly diminished myocardin expression (brown nuclear stain) in the SMCs populating the internal carotid artery (ICA) (G and H) and aorta (Ao) (I and J) of P2 Myocd^F/F/Wnt1-Cre⁺ mutant mice (H and J) compared with a control Myocd^F/F littermate (G and I). Original magnification, ×300 (carotid); ×400 (aorta).

Figure 3. Myocd^F/F/Wnt1-Cre⁺ mutant mice exhibit PDA.

(A) Intracardiac injection of toluidine blue reveals normal patterning of cardiac outflow tract and great arteries in a P2 Myocd^F/F control mouse. At P2 the DA is functionally occluded, giving rise to the ligamentum arteriosus (LA). RCA, right CA; LCA, left CA. (B) H&E-stained transverse section cut at the level of the AAo and DAo in a P2 Myocd^F/F control mouse, demonstrating occlusion of the DA and concomitant formation of the LA. Br, bronchus; Eso, esophagus. (C) Serial H&E-stained section cut at the level of the PA and DAo of a P2 Myocd^F/F control mouse. (D) H&E-stained caudal section cut at the level of the PA and DAo of a P2 Myocd^F/F control mouse. (E) Intra-injection of toluidine blue demonstrating widely PDA in P2 Myocd^F/F/Wnt1-Cre⁺ mutant mouse. (F) H&E-stained transverse section cut at the level of the AAo and DA of Myocd^F/F/Wnt1-Cre⁺ mutant mouse demonstrating widely PDA. (G) Serial H&E-stained more caudally cut section at the level of the AAo and DA of Myocd^F/F/Wnt1-Cre⁺ mutant mouse demonstrating widely PDA. (H) Serial H&E-stained caudal section cut at the level of the PA and DA of Myocd^F/F/Wnt1-Cre⁺ mutant mouse demonstrating PDA. Original magnification, ×10 (A and E); ×200 (B–D and F–H).

Figure 4. SMCs populating the DA of Myocd conditional mutant mice exhibit markedly diminished expression of SMC contractile proteins.

(A and B) H&E-stained section of the DA of Myocd^F/F control mouse (A) and Myocd^F/F/Wnt1-Cre⁺ mutant mouse (B). (C, E, and G) Immunohistochemical analyses of the DA of E16.5 Myocd^F/F control mouse demonstrating robust expression of SMA (green staining) (C), SM-MyHC (red staining) (E), and SM22α (green staining) (G). (D, F, and H) By contrast, expression of SMA (D), SM-MyHC (F), and SM22α (H) is markedly diminished in the DA of E16.5 Myocd^F/F/Wnt1-Cre⁺ mutant mice. (I and J) X-gal–stained section of P2 Myocd^F/F/Wnt1-Cre⁺/R26R⁺ triple mutant mice revealed that essentially all SMCs populating the DA express β-galactosidase (blue stain), confirming that these cells are of neural crest origin. Original magnification, ×100 (A–I); ×200 (J).

Figure 5. Myocd^F/F/Wnt1-Cre⁺ mutant mice exhibit a generalized defect in vascular SMC differentiation.

(A, D, G, and J) Serial histological sections of the AAo of an E16.5 Myocd^F/F control mouse. (A) H&E-stained AAo. (D, G, and J) Robust expression of SMA (D), SM-MyHC (G), and SM22α (J) is observed. (B, E, H, and K) Serial histological sections showing the AAo of an E16.5 Myocd^F/F/Wnt1-Cre⁺ mutant mouse. (B) H&E-stained AAo. (E, H, and K) Expression of SMA (E), SM-MyHC (H), and SM22α (K) is markedly diminished in the AAo of Myocd^F/F/Wnt1-Cre⁺ mutant mouse. (C, F, I, and L) Serial histological sections showing the DAo of an E16.5 Myocd^F/F/Wnt1-Cre⁺ mutant mouse. (C) H&E-stained section of the DAo. (F, I, and L) Robust expression of SMA (F), SM-MyHC (I), and SM22α (L) is observed in the DAo of a Myocd^F/F/Wnt1-Cre⁺ mutant mouse. Original magnification, ×200. (M) RT-PCR analysis of SMC contractile gene expression in Myocd^F/F/Wnt1-Cre⁺ mutant and control mice. mRNA harvested from the proximal aorta, carotid arteries, and DA of 4 P2 Myocd^F/F/Wnt1-Cre⁺ mice and 4 control mice was analyzed for expression of GAPDH, SMA, calponin-h1 (calponin), SM-MyHC, and SM22α by real-time RT-PCR as described (28). Data are expressed as mean gene expression (arbitrary units) ± SEM. Gray bars represent gene expression in Myocd^F/F/Wnt1-Cre⁺ mutant mice and black bars represent gene expression in control mice.

Figure 6. Ultrastructural changes in vascular SMCs populating the DA of Myocd^F/F/Wnt1-Cre⁺ mutant mice.

The DA of P2 Myocd^F/F control (A, C, E, and G) and Myocd^F/F/Wnt1-Cre⁺ mutant (B, D, F, and H) mice (n = 3 of each) were isolated, fixed, stained, and analyzed by electron microscopic analyses. (A and B) Low-power view demonstrating luminal occlusion of the DA in a control mouse (A), but widely PDA in the Myocd^F/F/Wnt1-Cre⁺ mutant mouse (E). Original magnification, ×500. (C) The tunica media of the DA in control mice exhibits regular circumferential organization with typical spindle-like morphology of SMCs. (D) By contrast, there is a loss of SMC mass with marked variability in SMC size and morphology as well as a concomitant increase in ECM in the mutant DA. Original magnification, ×25,000. (E) SMC morphology and ultrastructure in control Myocd^F/F DA demonstrating abundant myofibrils. Original magnification, ×25,000. (F) SMC morphology and ultrastructure in Myocd^F/F/Wnt1-Cre⁺ mutant DA. There is a marked decrease in myofibrils (large arrows) and a concomitant increase in synthetic organelles including rough endoplasmic reticulum (small arrows). Original magnification, ×100,000. (G and H) Comparison of SMC morphology in control (G) and mutant (H) DA demonstrating a relative increase in organelles associated with synthetic functions, including rough endoplasmic reticulum (arrows) and vacuoles (V) in Myocd^F/F/Wnt1-Cre⁺ mutant SMC (H) compared with the control SMC (G). m, mitochondria; n, nucleus. Original magnification, ×100,000.

Figure 7. Accumulation of ECM protein in the DA of P2 Myocd^F/F/Wnt1-Cre⁺ mutant mice.

Sections of the DA were stained with antibodies that recognize fibronectin (FN) and laminin and visualized by confocal microscopy. A dramatic increase in fibronectin expression was observed in the DA of Myocd^F/F/Wnt1-Cre⁺ mutants compared with control mice (compare A and B). In addition, a marked increase in expression of laminin was observed in the DA of myocardin conditional mutant mice (compare C and D). Original magnification, ×200.

Figure 8. Myocardin-regulated maintenance of the contractile SMC phenotype.

Primary SMCs isolated from Myocd^F/F aortae were infected with Ad-GFP (GFP) (A–C, G–I, and M–O) or Ad-Cre recombinase (Cre) (D–F, J–L, and P–R). Six days after infection, SMCs were immunostained with antibodies that recognize GFP, Cre, SM-MyHC (B, C, E, and F), calponin-h1 (H, I, K, and L), and SM22α (N, O, Q, and R), respectively. SMCs transduced with GFP and Cre stain green, while SMC contractile proteins stains red. (A–C) Colocalization of GFP and MyHC is observed. (D–F) By contrast, SMCs transduced with Ad-Cre (green) do not stain red indicating decreased SM-MyHC expression. (G–I) Colocalization of GFP and calponin is observed. (J–L) SMCs transduced with Ad-Cre do not stain red indicating decreased calponin expression. (M–O) Colocalization of GFP and SM22 is observed. SMCs transduced with Ad-Cre do not stain red indicating decreased SM22α expression. Original magnification, ×200. (S) Western blot analysis of cell lysates harvested from Myocd^F/F SMCs transduced with Ad-Cre and Ad-GFP. Abundant GFP and Cre are observed in cells transduced with Ad-GFP and Ad-Cre, respectively (rows 1 and 2). Expression of SMA and SM22α was dramatically decreased in Myocd^F/F SMCs transduced with Ad-Cre relative to levels observed in cells transduced with Ad-GFP (lanes 3 and 4). Expression of GAPDH was comparable in SMCs transduced with Ad-Cre and Ad-GFP (lane 5).