Resistance of Acta2 mice to aortic disease is associated with defective release of mutant smooth muscle α-actin from the chaperonin-containing TCP1 folding complex

Figures

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  Figure 1. Force development is decreased in aortic tissues from Acta2+/R149Cmice.A, two-dimensional gel of lysates of WT and Acta2R149C/+ aorta with pan-actin antibody confirms the production of R149C SM α-actin in the aorta of Acta2R149C/+ mice at 2 months of age (n = 3 mice per genotype). B, Western blot analysis of aortic tissue lysates shows increased amounts of smooth muscle myosin heavy chain (SM MHC) but decreased amounts of calponin, whereas there was no change in SM22α and SM α-actin in the aortas of Acta2R149C/+ mice compared with WT mice at 2 months of age (n = 6 per genotype). C, KCl (65 mM) or phenylephrine (PE, 10 μM)-induced force generation of aortic rings from both ascending (ASC) and descending (DSC) thoracic aorta of Acta2R149C/+ mice was significantly decreased during the initial (1 min) or sustained (5 min) contraction phases compared with the WT mice at 2 months of age. Force measurements were normalized as grams of developed force per tissue wet weight. ∗∗p < 0.01, compared with WT mice. n = 5 for ASC and 10 for DSC. D, analysis of aortic tissue lysates showed that Acta2R149C/+ and WT mice at 2 months of age had comparable extents of RLC phosphorylation at rest…

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  Figure 2. Acta2R149C/+mice have increased aortic wall thickness but do not develop thoracic aortic aneurysms, even when blood pressure is increased.A, echocardiography analysis shows smaller size of ascending aorta and aortic root in the Acta2R149C/+ mice compared with WT mice in young mice, but after 30 weeks of age, the mutant and WT aortas are of the same diameter and remain similar through 104 weeks of age (p < 0.05, p < 0.01 compared with WT mice; n = 7 or 8 per group). B, histologic stains of aortic sections showed significantly increased aortic wall thickness with significant decrease in cell density, but there was no significant increase in elastin layers and elastin breaks in the Acta2R149C/+ mice at 5 months of age (p < 0.05, p < 0.01 compared with WT mice; n = 5 per group). Movat and H&E scale is 100 μm. VVG scale is 75 μm. C, weekly echocardiograms indicated that Acta2R149C/+ mice subjected to transaortic constriction (TAC) at 3 months of age had similar growth rate of both ascending aorta and aortic root to WT mice (n = 3 or 6 per group). D, echocardiography analysis demonstrated no aortic enlargement in Acta2R149C/+ mice compared with WT mice when blood pressure was elevated…

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  Figure 3. Polymerization and motility of WT and mutant actins and their interaction with CCT and actin-binding proteins.A, quantitation of in vitro translation reaction products shows significantly increased ratio of CCT-bound:released actin for R149C actin compared with WT actin (1.14 ± 0.47 versus 0.42 ± 0.1, p = 0.01) and R149C/N299T actin (0.64 ± 0.22, p = 0.048). Representative gel shown from six different assays. B, the actin concentration dependence of the polymerization rate of WT, N299T, and R149C/N299T monomers. See Table 2 for assembly and disassembly values. C, the polymerization rate of R149C/N299T in the absence or the presence of 3 μM profilin yielded a dissociation constant of the mutant actin for profilin of 2.7 μM, comparable to the value of 3.0 μM previously obtained for WT (7). The curve is the fit to the data as described previously (7). D, the actin polymerization rate of WT and R149C/N299T in the presence of 3 μM MRTF-A. The curves are fits to the data as described previously (8). Fit to the R149C/N299T data yields a Kd of 3.3 μM and a Hill coefficient of 4.7, versus WT values of 1.8 μM and a Hill coefficient of 4.7. All WT data (B–D) are from previously published…

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  Figure 4. Characterization of WT and Acta2R149C/+cells. SMCs were explanted from the ascending aorta of WT and Acta2R149C/+ mice. A, immunoblot analyses of contractile proteins showed similar amount of SM22α and SM α-actin but increased amounts of SM MHC in Acta2R149C/+ SMCs. Scale bars represent 25 μm. B, ultracentrifugation was used to separate F-actin (pellet) from G-actin (supernatant). There was no difference in F-actin (polymerized) with little or no G-actin (monomeric) in both mutant and WT SMCs. C, immunofluorescent staining of SMCs using a SM α-actin antibody showed that Acta2R149C/+ SMCs assemble SM α-actin filaments that were longer and less robust than WT SMCs. D, STORM images of cells expressing WT (left) or R149C (right) actin. E, left, the Fourier transform of the images showed many high-frequency bands for the WT cell. Right, histogram of the distance of each actin molecule's closest neighbor in WT and R149C cells. The solid lines are the sum of two Gaussian peaks for WT (black) and R149C (blue). The dashed line shows individual Gaussian peaks for WT (3.5 ± 2.2 and 6.4 ± 4.6 nm) and R149C (4.1 ± 3.0 and 7.7 ± 5.5 nm). F, the CCT complex was immunoprecipitated using…