Nuclear Smooth Muscle α-actin in Vascular Smooth Muscle Cell Differentiation

Summary Missense variants throughout ACTA2, encoding smooth muscle α-actin (αSMA), predispose to adult onset thoracic aortic disease, but variants disrupting arginine 179 (R179) lead to Smooth Muscle Dysfunction Syndrome (SMDS) characterized by childhood-onset diverse vascular diseases. Our data indicate that αSMA localizes to the nucleus in wildtype (WT) smooth muscle cells (SMCs), enriches in the nucleus with SMC differentiation, and associates with chromatin remodeling complexes and SMC contractile gene promotors, and the ACTA2 p.R179 variant decreases nuclear localization of αSMA. SMCs explanted from a SMC-specific conditional knockin mouse model, Acta2SMC-R179/+, are less differentiated than WT SMCs, both in vitro and in vivo, and have global changes in chromatin accessibility. Induced pluripotent stem cells from patients with ACTA2 p.R179 variants fail to fully differentiate from neural crest cells to SMCs, and single cell transcriptomic analyses of an ACTA2 p.R179H patient’s aortic tissue shows increased SMC plasticity. Thus, nuclear αSMA participates in SMC differentiation and loss of this nuclear activity occurs with ACTA2 p.R179 pathogenic variants.


Introduction 1
Heterozygous missense mutations in ACTA2, the gene encoding smooth muscle 2 specific alpha-actin (αSMA), cause a diverse spectrum of vascular diseases, including 3 thoracic aortic disease, premature coronary artery disease, and moyamoya-like 4 cerebrovascular disease. 1, 2 All identified ACTA2 pathogenic variants cause thoracic 5 aortic disease, likely due to the loss of smooth muscle cell (SMC) contraction, resulting 6 in compensatory signaling by SMCs to correct for deficient homeostatic force 7 generation. 3 Only a subset of missense pathogenic variants in ACTA2 are associated with 8 non-thoracic aortic vascular diseases, and clinical data confirm that specific ACTA2 9 missense variants are associated with a risk for either early onset coronary artery disease 10 or moyamoya-like cerebrovascular disease. 1 These significant genotype to phenotype 11 correlations suggest ACTA2 pathogenic variants alter αSMA by distinct mechanisms that 12 have disparate effects on SMC phenotype. 13 De novo pathogenic variants that disrupt arginine 179 (R179), lead to a severe, 14  SM22α when compared with cells expressing WT αSMA (Fig. 3A, Supplemental Fig.  1 VIB). Co-immunoprecipitation with antibodies directed against either INO80 or BRG1 2 indicate decreased association of R179C αSMA with chromatin remodeling complexes 3 compared with WT αSMA (Fig. 3B,C). By contrast, more β-actin is co-precipitated with 4 INO80 in the cells expressing R179C mutant αSMA compared with WT (Fig. 3B). 5 Finally, ChIP pulldowns with αSMA and β-actin antibodies confirm decreased binding of 6 R179C mutant αSMA to the promoter regions of SMC-specific genes, while β-actin 7 binding is increased in the cells expressing mutant compared with WT αSMA (Fig. 3D). 8 There are no significant differences in binding of αSMA on the Klf4 promoter, suggesting 9 a specific function of αSMA on SMC-specific gene promoters. To further assess whether 10 loss of nuclear αSMA has a functional impact, ChIP pulldowns were performed with 11 antibodies against a histone marker of transcriptional activation, H3K4me3, and a marker 12 of transcriptional repression, H3K27me3. The CArG box regions of SMC-specific genes 13 have significantly decreased H3K4me3 in cells expressing R179C mutant αSMA 14 compared with WT αSMA ( Figure 3E). By contrast, there is no change in these histone 15 modifications on the Klf4 promoter. Taken together, these results suggest that arginine 16 179 alteration disrupts the nuclear localization and function of αSMA, and that this loss 17 of nuclear function leads to changes in SMC-associated genomic loci. We generated a SMC-specific Acta2 R179C knock-in mouse model (termed 22 Acta2 SMC-R179C/+ ) and validated that the mutation was present in 66% of SMCs in the 23 aortic tissue in vivo, but SMCs explanted from the ascending aorta are a pure 1 heterozygous population by RNA sequencing and 2D gel analyses. 15 These findings 2 suggest that the Acta2 SMC-R179C/+ SMCs proliferate more rapidly than the WT SMCs, and 3 we found that both proliferation and migration were increased in the mutant SMCs when 4 compared to SMC explanted from WT mouse ascending aortas (Fig. 4A,B). Acta2 SMC-5 R179C/+ SMCs also have significantly reduced expression and levels of contractile proteins, 6 Cnn1, Tagln, and Myh11 (Fig. 4C) and increased expression of pluripotency markers 7 Nanog, Klf4, Oct4, and Sox2 when compared with WT SMCs (Fig. 4D). Please note that 8 interpretation of Oct4 expression may be complicated by the existence of pseudogenes. 16

9
Protein levels of calponin, SM22α, and SMMHC are decreased in Acta2 SMC-R179C/+ SMCs 10 consistent with the reduced RNA expression (Fig. 4E, Supplemental Fig. VIIA). 11 Acta2 SMC-R179C/+ SMCs have reduced levels of the myocardin related transcription factor 12 A (Mkl1), a cofactor that binds to serum response factor (SRF) to drive contractile gene 13 expression (Fig. 4E, Supplemental Fig. VIIA). Since a highly migratory behavior is a 14 hallmark of neural crest cell (NCC) progenitors, 17 the less differentiated phenotype of 15 Acta2 SMC-R179C/+ SMCs may represent failure of NCCs to completely differentiate into 16 SMCs during development rather than phenotypic modulation of differentiated SMCs. 17 Acta2 SMC-R179C/+ SMCs have significantly decreased accumulation of αSMA in the 18 nucleus and concomitant increased cytosolic accumulation. TGFβ1 treatment increases 19 αSMA levels in both WT and Acta2 SMC-R179C/+ SMCs (Fig. 4F, Supplemental Fig. VIIB). 20 WT SMCs have nuclear αSMA and β-actin present with or without latrunculin A 21 treatment, whereas β-actin in the nuclear fraction of Acta2 SMC-R179C/+ SMCs decreases 22 further with latrunculin A treatment (Fig. 4G, Supplemental Fig. VIIC). Immunostaining 23 of αSMA in WT and mutant SMC nuclei followed by quantitation of fluorescence 1 intensity confirms reduced nuclear αSMA in Acta2 SMC-R179C/+ SMCs (Fig. 4H,I). To 2 determine if association of αSMA with chromatin remodeling complexes is altered in 3 Acta2 SMC-R179C/+ SMCs, co-immunoprecipitation with antibodies directed against either 4 INO80 or BRG1 were pursued. Co-precipitation of αSMA with chromatin remodeling 5 complexes was decreased in Acta2 SMC-R179C/+ SMCs, as was co-precipitation of β-actin, 6 although TGFβ1 treatment rescues actin interactions with these complexes (Fig. 4J, 7 The R179C mutation causes significant disruption of polymerization of αSMA in 9 addition to the loss of nuclear function described here. 18 To confirm that the decreased 10 differentiation of Acta2 SMC-R179C/+ SMCs is not due to cytosolic actin disruptions, WT 11 mouse SMCs were treated with an αSMA disrupting peptide (SMAfp) or with a peptide 12 designed to disrupt skeletal α-actin (SKAfp) as a control. These peptides have been 13 previously characterized, 19 and we previously showed that treatment with SMAfp 14 marginally increases SMC proliferation through increased expression of PDGFRβ. 20 15 Here, cells were treated with 5 μg/mL SMAfp to completely disrupt αSMA filaments, 16 while SKAfp moderately affects αSMA filament formation (Fig. 4K

Mouse SMCs with knock-in R179C mutation have altered chromatin accessibility 2
To determine if decreased nuclear actin in Acta2 SMC-R179C/+ SMCs alters global 3 chromatin remodeling, assay for transposase-accessible chromatic (ATAC)-sequencing 4 was pursued in WT and Acta2 SMC-R179C/+ SMCs. We identified 2466 peak regions with 5 greater than 1.5-fold differential accessibility, including 1018 peaks with increased 6 accessibility and 1448 peaks with decreased accessibility in Acta2 SMC-R179C/+ SMCs. 7 Integrated peak region-gene association calls and pathway analysis using GREAT were 8 performed (Supplemental Fig. XA,B). GO term analysis of the genes associated with 9 peaks of decreased accessibility in Acta2 SMC-R179C/+ SMCs shows enrichment of multiple 10 biological processes related to muscle and cardiac cell development and contraction, 11 consistent with the lack of differentiation observed in these SMCs (Fig. 5A). In contrast, 12 GO terms associated with regions of increased accessibility in Acta2 SMC-R179C/+ SMCs 13 include terms related to cortical actin cytoskeleton or actomyosin structure organization, 14 which are terms associated with the cortical actin rearrangements necessary for cell 15 migration (Fig. 5B). These differences in chromatin accessibility align with differences in 16 gene expression and cellular behavior in Acta2 SMC-R179C/+ SMCs, providing evidence that 17 chromatin remodeling changes due to loss of nuclear actin in Acta2 SMC-R179C/+ SMCs may 18 underlie the lack of differentiation and maintenance of some NCC phenotypic features. 19 20

Mouse SMCs with knock-in R179C mutation are less differentiated in vivo 21
Acta2 SMC-R179C/+ mice are mosaic with knock-in of the R179C variant in 22 approximately 67% of SMCs based on single cell RNA-sequencing (scRNA-seq) of 23 aortic and carotid artery tissue from these mice. 15 Transcriptomic data from WT and 1 Acta2 SMC-R179C/+ aortic cells visualized in UMAP space identified two distinct clusters of 2 SMCs in Acta2 SMC-R179C/+ mice, one of which overlapped with the single SMC cluster in 3 WT mice (Fig. 5C). Based on analysis of the transcriptomic data, the "SMC1" cluster 4 represents cells without the R179C variant and the "SMC2" cluster represents cells with 5 the variant. Data from cells in the SMC clusters from WT and Acta2 SMC-R179C/+ tissue 6 were assessed for differentially expressed genes (DEGs), and 289 DEGs were identified, 7 with 122 genes downregulated and 167 genes upregulated in SMC2 cluster when 8 compared with WT SMCs and SMC1 clusters (Fig. 5D). GO term enrichment analysis 9 identified 10 terms significantly upregulated in SMC2, including regulation of cell 10 proliferation (Fig. 5E). To visualize these changes in cell proliferation, we combined all 11 genes represented in GO term 0008283 (Cell population proliferation) and visualized the 12 decreasing its binding to SRF in the nucleus and reducing SRF-driven expression of SMC 18 differentiation markers. 14 To determine whether this pathway contributes to de-19 differentiation of ACTA2 p.R179 cells, actin polymerization was assessed by an 20 ultracentrifugation-based F/G actin assay. Cells derived from an SMDS patient had no 21 change in F to G actin ratio compared with control cells (Fig. 6G). ACTA2 p. R179C 22 SMCs have a significant increase in the ratio of nuclear to cytosolic MKL1 compared 23 with controls ( Fig. 6H,I). These results suggest that de-differentiation of the mutant cells 1 is not the result of the MKL1/SRF axis. 2 To accurately assess whether loss of nuclear αSMA prevents complete 3 differentiation of SMCs, Crispr/Cas9 genomic editing was used to induce a homozygous 4 loss-of-function allele in ACTA2 in control iPSCs. These cells were differentiated into 5 NEPCs and then SMCs, and the cells with near-total loss of ACTA2 expression also had 6 decreased expression of other SMC contractile genes (Fig. 6J) and increased expression 7 of pluripotency-associated genes ( Fig. 6K) comparable to cells derived from ACTA2 8 p.R179 patients. These results further support that loss of nuclear αSMA prevents the 9 complete differentiation of SMCs. 10 11

Single cell transcriptomics of the aorta of patient with ACTA2 p.R179H confirms 12 dedifferentiation in vivo 13
To determine the functional consequences of ACTA2 p.R179 variants in human 14 aortic disease in vivo, the ascending aorta from an 8-year-old child with SMDS due to a 15 de novo ACTA2 c.536G>A variant (p.R179H) was assessed using single cell 16 transcriptomics, along with a 12mm diameter distal ascending aortic tissue sample from a 17 2-year-old heart donor as a control (Fig. 7A,B). The patient had classic features of SMDS 18 including congenital mydriasis, intestinal malrotation, repair of a patent ductus arteriosus 19 at two weeks of age, and progressive aneurysmal enlargement of the root and ascending 20 aorta (37mm diameter, Z-score 11.0 at the time of surgery). 4 We performed enzymatic 21 digestion and single-cell RNA sequencing (scRNA-seq) on fresh aneurysm tissue at the 22 time of elective aortic repair surgery using a validated protocol. 25, 26 Following single cell 23 captures and mRNA library preparation, samples were sequenced and integrated into a 1 joint dataset (6,263 cells) using standard workflows within the Seurat package in R (Fig.  2   7C). 27, 28 Low-resolution clustering of the integrated dataset identified 10 cell types, 3 including SMC, fibroblast, endothelial (EC), and macrophage clusters (Fig. 7D) Progressively reduced expression of these markers correlates with multiple distinct 2 subsets within the broader SMC dataset populated almost entirely by ACTA2 p.R179H 3 cells. Although expression of some stem cell markers was not present in these subsets 4 (e.g., OCT4, SOX2), there are uniquely activated markers in these phenotypic offshoots.  We hypothesize that decreased levels of αSMA in the nucleus underlie the lack of 1 differentiation in heterozygous ACTA2 R179 SMCs. Treatment with TGFβ1 increases 2 αSMA nuclear localization in the R179 mutant cells and partially rescues the decreased 3 differentiation. However, the rescue of SMC contractile protein levels is incomplete, and 4 ACTA2 p.R179 cells undergo longer-term treatment with TGFβ1 during NEPC to SMC 5 differentiation and still fail to fully differentiate. These data suggest the possibility that 6 R179 mutant αSMA has functional defects in the nucleus even when nuclear levels are 7 increased by TGFβ1 treatment, and future studies will address this possibility. SMCs that are dedifferentiated with increased chondrogenic gene expression, a similar 1 transcriptional profile to the chondromyocyte cluster in the ACTA2 p.R179H aortic tissue, 2 supporting the conclusion that these cells are also SMC-derived. 33 Interestingly, we found 3 that multiple gene targets of EZH2 are upregulated in the ACTA2 p.R179H tissue. A 4 recent paper showed in the absence of β-actin, BRG1 genomic association is globally 5 depleted, leading to increased EZH2 recruitment, and in this context EZH2 acts as a 6 transcriptional activator of a subset of genes. 34 EZH2 in the cytosol has also been shown 7 to regulate actin polymerization, 35 and it has been speculated that nuclear EZH2 could 8 similarly regulate nucleoskeletal assembly of actin filaments. 36 EZH2 is a 9 methyltransferase responsible for trimethylation of H3K27. 36 H3K27me3 mark at the 10 Tagln and Cnn1 loci is increased in SMCs expressing R179C αSMA, suggesting there 11 may be increased EZH2 activity in cultured cells as well as the aortic tissue.

pathogenesis. 2
Previous data addressing the role of nuclear β-actin in cellular differentiation 3 support that efflux of nuclear β-actin during development is a mechanism of cell fate 4 transition. Importantly, we observed decreased nuclear β-actin in NEPCs relative to 5 differentiated SMCs (Fig. 1G). In Xenopus oocytes, high levels of nuclear β-actin are 6 present due to low expression of its nuclear exporter, Xpo6. As oocytes differentiate, 7 Xpo6 expression is increased, β-actin is exported from the nucleus, and differentiation is analysis of the ATAC-seq data was "vascular development", which potentially supports 19 our hypothesis that nuclear αSMA preferentially activates genes critical for SMC 20 differentiation over β-actin. Our own ATAC-seq analysis further supports our hypothesis 21 by identifying that, in Acta2 SMC-R179C/+ SMCs with less nuclear αSMA, genes associated 22 with cortical actin rearrangements needed for cellular migration are more accessible and 23 genes associated with muscle differentiation and contraction are less accessible. In 1 summary, these studies all indicate that nuclear actin is an epigenetic remodeling factor 2 that helps coordinate chromatin accessibility and gene expression and establish cell fate, 3 and our data support that αSMA, rather than β-actin, coordinates these activities in the 4 nucleus of developing SMCs. The mechanism preventing nuclear localization of R179 mutant αSMA is 1 unidentified. In Acta2 SMC-R179C/+ SMCs, there is a greater than 50% reduction in nuclear 2 αSMA in heterozygous cells, indicating a dominant negative effect of mutant αSMA on 3 WT αSMA nuclear localization. We have previously reported that ACTA2 pathogenic 4 variants disrupting arginine 258 also predispose to moyamoya-like cerebrovascular 5 disease, 1 and found that methylation at the corresponding amino acid in yeast actin or 6 human β-actin (R256) is required for nuclear function. 12 Arginine methylation is a post-7 translational modification linked with nuclear functions of proteins, including chromatin 8 organization, gene expression, and RNA processing. 56 Methylation of yeast actin at R177 9 does occur; 12 and, in the context of missense mutations affecting R179, loss of 10 methylation would potentially disrupt nuclear function of the protein. Interestingly, a 11 second band for nuclear αSMA migrating at a slightly higher molecular weight was noted 12 on immunoblots in control NEPCs (Fig. 1G), raising the possibility that additional post-13 translational modifications of αSMA could be important for its nuclear function. 14 Alternatively, R179 mutant αSMA could be excluded from the nucleus due to aberrant 15 protein interactions, including altered interactions with proteins responsible for importing 16 and exporting αSMA to the nucleus. β-actin binds to cofilin and is then imported by Ipo9, 17 while profilin-bound actin is exported by Xpo6. 54, 57 In vitro studies previously showed 18 that both R179 and R258 mutant αSMA bind more tightly to profilin, 18, 58 and we found 19 less nuclear profilin in cells expressing R179 mutant αSMA compared with WT ( Fig. 3A  20 and data not shown). Future studies will focus on profilin or additional binding proteins 21 to explore the mechanism excluding R179 mutant αSMA from the nucleus. 22 This work establishes a novel and critical role for αSMA as an epigenetic factor 1 involved in the developmental specification of SMCs. We further hypothesize that 2 cardiac and skeletal muscle-specific α-actins play a similar role by accumulating in the 3 nucleus during development and guiding myocyte-specific chromatin remodeling and 4 fate specification. Loss of nuclear αSMA with ACTA2 R179 mutation causes alterations 5 in chromatin accessibility, leading to incomplete differentiation of SMCs. The 6 consequence of this incomplete differentiation is retention of stem cell-like phenotypes of 7 increased proliferation and migration, which may contribute to the occlusive vascular 8 disease in SMDS and multiple trajectories of SMC modulation. Chaponnier for their generosity in sharing the SKAfp and SMAfp peptides for this study.

Declaration of Interests 22
The authors declare no competing interests.