Smooth muscle α-actin missense variant promotes atherosclerosis through modulation of intracellular cholesterol in smooth muscle cells

Figures

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  Figure 1. Acta2R149C/+Apoe−/− mice have increased atherosclerotic plaque burden. (A, B) Oil Red O staining shows Acta2R149C/+Apoe−/− mice have increased atherosclerotic plaque burden in the whole, thoracic, and abdominal aortas, but not in the ascending aortas compared to Apoe−/− mice (n = 7 per genotype per sex, unpaired student’s t-test followed by Welch’s correction) after 12 weeks of HFD. (C, D) Plaque burden is greater in the roots and ascending aortas of Acta2R149C/+Apoe−/− mice after 6 weeks of HFD (N = 5 per genotype per sex, Mann–Whitney test), (C), and in the same aortic regions of Acta2R149C/+ mice after 12 weeks of HFD, (N = 5 per genotype per sex, unpaired student’s t-test followed by Welch’s correction), (D). (E) Medial layers of both the roots and ascending aortas of Acta2R149C/+Apoe−/− mice had significantly fewer tdTomato+ cells than those of Apoe−/− mice. (F) Cell density in the medial layers of the ascending aortas but not in the aortic roots is significantly lower in Acta2R149C/+Apoe−/− mice than Apoe−/− mice (N = 9, unpaired student’s t-test followed by Welch’s correction and Mann–Whitney test, respectively). Error bars represent standard deviation. *, **, ***, ****—P…

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  Figure 2. Single-cell transcriptomic analyses of Acta2R149C/+Apoe−/− and Apoe−/− aortas: (A). Uniform manifold approximation and projection (UMAP) visualization of all cell clusters obtained after scRNA-seq performed on tissue from the aortic root to the distal aortic arch of age-matched male SMC lineage-traced Acta2R149C/+Apoe−/− and Apoe−/− mice (N = 4). (B) The distribution of SMC lineage reporter tdT expression across all cell clusters. (C) UMAP visualization and further clustering of SMCs into subtypes. (D) Top genetic markers identified for each subtype of SMCs. (E) Contractile SMCs show the highest difference in transcriptomic profiles between Acta2R149C/+Apoe−/− and Apoe−/− mice when perturbations induced by a single point mutation in the Acta2 allele were quantified. (F) Differentially expressed genes across all subtypes of SMCs between the two genotypes. P-values used to determine statistical significance are all two-sided. cSMC—contractile SMC, mSMC—modulated SMC.

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  Figure 3. Augmented ER stress and phenotypic modulation in Acta2R149C/+ SMCs. (A, B) Increased Atf4 and Klf4 expression and KLF4 luciferase activity (A), and increased levels of eIF2α phosphorylation, ATF4 and KLF4, (B), show baseline activation of PERK signaling in Acta2R149C/+ SMCs. (C) Increased splicing of Xbp1 (sXbp1/uXbp1) and ATF6 luciferase activity demonstrate activation of IRE1α and ATF6 signaling, respectively, at baseline in Acta2R149C/+ SMCs. (D) cSMCs, as well as both mSMC clusters in Acta2R149C/+Apoe−/− aortas showed significantly higher PERK signaling signature compared to Apoe−/− aortas. P-values used to determine statistical significance are all two-sided. (E) Baseline and MBD-Chol-driven increases in Atf4 and Klf4 expression, are reversed by treatment with the PERK pathway inhibitor ISRIB. Expression of Cnn1 is decreased and modulation markers Lgals3 and Spp1 are increased either at baseline or with exposure to 2.5 µg/mL MBD-Chol in Acta2R149C/+ SMCs, compared to with 10 µg/mL MBD-Chol in WT SMCs. These changes are reversed with ISRIB. (F) Acta2R149C/+ SMCs showed both increased proliferation by EdU incorporation and migration by transwell migration assay at baseline and…

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  Figure 4. Increased HSF1 activation drives PERK signaling in Acta2R149C/+ SMCs. (A) Acta2R149C/+ SMCs exhibit increased levels of total and phosphorylated HSF1 (p-HSF1) at baseline. (B) HSF1 luciferase activity and expression of HSF1 target Hspa1a are also upregulated at baseline in Acta2R149C/+ SMCs. (C) Single-cell transcriptomics reveal increased HSF1 activation gene expression signature and decreased HSF1 inhibition signature in the cSMC cluster of Acta2R149C/+Apoe−/− aortas. P-values used to determine statistical significance are all two-sided. (D) In Acta2R149C/+ SMCs, siRNA-mediated depletion of Hsf1 reduces activation of the PERK pathway at baseline (Atf4 and Klf4 expression, along with KLF4 luciferase activity) as well as decreasing phenotypic modulation (increases Cnn1 expression and reduces Lgals3 and Spp1 expression). (E) Heatshock of WT SMCs increases HSF1 luciferase activity and expression of Hsf1 and its target Hsp90ab1, while simultaneously activating the PERK pathway (Atf4 and Klf4 expression and KLF4 luciferase activity) and inducing phenotypic modulation (decreased expression of Cnn1 and increased expression of Lgals3 and Spp1). All cellular data with cholesterol were…

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  Figure 5. HSF1 activation in Acta2R149C/+ SMCs increases HMG-CoA reductase levels. (A–C) Hmgcr expression is significantly elevated in Acta2R149C/+ SMCs (A), along with HMG-CoAR enzymatic activity and cholesteryl ester (CE) levels (B), which are both significantly reduced following siRNA-mediated depletion of Hsf1 (C). (D) Heat shocking WT SMCs also induces Hmgcr expression. (E) Treatment with the HMG-CoAR inhibitor pravastatin reduces baseline activation of the PERK pathway (Atf4 and Klf4 expression and KLF4 luciferase activity) and phenotypic modulation (decreased expression of Cnn1 and increased expression of Lgals3). (F, G) Treatment with pravastatin reduced plaque burden to similar levels in Acta2R149C/+Apoe−/− and Apoe−/− mice (N = 8 males). (H, I) SMCs explanted from an ACTA2 p.R149H patient demonstrated increased HSF1 and KLF4 activation at baseline, as well as increased expression of HMGCR and elevated levels of cholesteryl esters. Two-group comparisons were performed using unpaired student’s t-test, followed by Welch’s correction. Multiple group comparisons for both cellular and animal data were analyzed by 2-way ANOVA followed by Tukey’s multiple comparisons test. Error bars…

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  Figure 6. Misfolding of the R149C SMA triggers HSF1 activation. (A, B) Despite expressing at similar transcript levels when expressed from a lentiviral construct in Acta2−/− SMCs (A), the R149C SMA monomer is not detectable on an immunoblot, while the WT, N299T, and R149C; N299T SMA monomers are (B). (C) Expression of R149C SMA leads to increased HSF1 activity, increased levels of cholesteryl esters, induction of Hmgcr expression and activity, and elevated KLF4 luciferase activity, which are all reversed by addition of the second point mutation, N299T. (D) Addition of the N299T mutation reverses the 2.5 µg/mL MBD-Chol-induced increased PERK activation (Atf4 and Klf4 levels) and augmented modulation (reduced Cnn1 expression and increased Lgals3 and Spp1 expression) caused by the R149C mutation alone. All cellular data were analyzed by 2-way ANOVA followed by Tukey’s multiple comparisons test. *, **, ***, ****—*P < 0.05, 0.01, 0.001, and 0.0001, respectively. CE—cholesteryl ester.