Nuclear smooth muscle α-actin participates in vascular smooth muscle cell differentiation

ABSTRACT Missense variants throughout _ACTA2_, encoding α-smooth muscle actin (αSMA), predispose to adult-onset thoracic aortic disease, but variants disrupting arginine 179 (R179) lead to

smooth muscle dysfunction syndrome characterized by diverse childhood-onset vascular diseases. Here we show that αSMA localizes to the nucleus in wild-type smooth muscle cells (SMCs),

enriches in the nucleus with SMC differentiation, and associates with chromatin remodeling complexes and SMC contractile gene promoters. The _ACTA2_ p.Arg179 αSMA variant shows decreased

nuclear localization. Primary SMCs from _Acta2_SMC-R179C/+ mice are less differentiated than wild-type SMCs in vitro and in vivo and have global changes in chromatin accessibility. Induced

pluripotent stem cells from participants with _ACTA2_ p.Arg179 variants fail to fully differentiate from neuroectodermal progenitor cells to SMCs, and single-cell transcriptomic analyses of

an _ACTA2_ p.Arg179His participant’s aortic tissue show increased SMC plasticity. Thus, nuclear αSMA participates in SMC differentiation, and loss of this nuclear activity occurs with

_ACTA2_ p.Arg179 pathogenic variants. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS INDUCIBLE PRMT1 ABLATION IN ADULT VASCULAR SMOOTH MUSCLE LEADS TO CONTRACTILE

DYSFUNCTION AND AORTIC DISSECTION Article Open access 11 October 2021 INTERLEUKIN-11 IS IMPORTANT FOR VASCULAR SMOOTH MUSCLE PHENOTYPIC SWITCHING AND AORTIC INFLAMMATION, FIBROSIS AND

REMODELING IN MOUSE MODELS Article Open access 20 October 2020 YY1 DIRECTLY INTERACTS WITH MYOCARDIN TO REPRESS THE TRIAD MYOCARDIN/SRF/CARG BOX-MEDIATED SMOOTH MUSCLE GENE TRANSCRIPTION

DURING SMOOTH MUSCLE PHENOTYPIC MODULATION Article Open access 11 December 2020 DATA AVAILABILITY scRNA-seq datasets generated for this manuscript are available in the GEO under accession

number GSE201091. The ATAC-seq dataset generated for this manuscript has been deposited in the GEO and is available under accession number GSE241055. All reagents and resources applicable to

this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper. CODE AVAILABILITY No novel code or algorithm was generated for

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  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by an America Heart Association Merit Award (to D.M.M.), the National Heart, Lung and Blood Institute (RO1

HL146583 to D.M.M.), the Olivia Petrera-Cohen Research Fund (to D.M.M.), the John Ritter Foundation (to D.M.M.), Marylin and Frederick R. Lummis, MD, Fellowship in the Biomedical Sciences

(to A.K.), National Institutes of Health (NIH) TL1TR003169 (to A.K.), NIH UL1TR003167 (to A.K.), NIH F32HL154681 (to A.J.P.), NIH R01HL157949 (to M.P.F.), British Heart Foundation awards

RG/17/5/32936 and FS/18/46/33663 (to S.S.) and American Heart Association grant 20CDA35310689 (to C.S.K.). The funders had no role in study design, data collection and analysis, decision to

publish or preparation of the manuscript. Confocal microscopy was performed at the Center for Advanced Microscopy, Department of Integrative Biology and Pharmacology at McGovern Medical

School, UTHealth. We thank G. Gabbiani and C. Chaponnier from the University of Geneva for their generosity in sharing the SKAfp and SMAfp peptides for this study. AUTHOR INFORMATION Author

notes * Shuangtao Ma Present address: Department of Medicine, Michigan State University, East Lansing, MI, USA AUTHORS AND AFFILIATIONS * Division of Medical Genetics, Department of Internal

Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, USA Callie S. Kwartler, Anita Kaw, Pujun Guan, Shuangtao Ma, Xue-yan Duan, Caroline

Kernell, Charis Wang, Jose Emiliano Esparza Pinelo, Mikayla S. Borthwick Bowen, Jiyuan Chen & Dianna M. Milewicz * Department of Cardiothoracic Surgery, Stanford University, Stanford,

CA, USA Albert J. Pedroza & Michael P. Fischbein * Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Yuan

Zhong * Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom Sanjay Sinha * Institute of Cancer Research, Shenzhen Bay Laboratory, Shenzhen, China

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CONTRIBUTIONS D.M.M. and C.S.K. designed the study. C.S.K. planned the individual experiments. C.S.K., A.K., S.M., X.y.D., C.K., J.E.E.P., M.S.B.B. and J.C. performed the cellular

experiments. A.K. and A.J.P. obtained the sample and analyzed the scRNA-seq on mouse tissue. A.J.P. and M.P.F. obtained the sample and analyzed the scRNA-seq on participant tissue. C.S.K.

and A.J.P. obtained the sample and analyzed the ATAC-seq on cultured SMCs. P.G. performed the integrated analysis combining scRNA-seq and ATAC-seq datasets. Y.Z. and X.S. consulted on

nuclear actin functions and contributed to the design of experiments. S.S. reprogrammed a participant stem cell line and assisted with the stem cell differentiation protocol. D.M.M. and

C.S.K. interpreted the data and drafted the manuscript. D.M.M. and M.P.F. obtained funding for this work. CORRESPONDING AUTHORS Correspondence to Callie S. Kwartler or Dianna M. Milewicz.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Cardiovascular Research_ thanks the anonymous reviewers for

their contribution to the peer review of this work. Primary Handling Editor: Vesna Todorovic, in collaboration with the _Nature Cardiovascular Research_ Team. ADDITIONAL INFORMATION

PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 ΑSMA LOCALIZES TO

THE NUCLEUS CONCURRENTLY WITH SMC DIFFERENTIATION. A) Quantitation of immunoblot in Fig. 1A shows that TGFβ1 stimulation for 48 h increases protein levels of αSMA in both the nucleus and

cytosol, while PDGF-BB treatment for 24 h does not affect αSMA nuclear localization (n=3 independent experiments). Significance was assessed by one-way ANOVA followed by Tukey’s multiple

comparisons test. B) Quantitation of the latrunculin (LtA) treatment immunoblot shown in Fig. 1C shows that depolymerization of actins by LtA treatment does not affect nuclear localization

of actin, supporting that the observed nuclear localization is accurate (n = 4 independent experiments). Significance was assessed by Kruskal-Wallis test with Dunn’s multiple comparisons

test. C) Quantitation of the NEPC to SMC differentiation timecourse immunoblot shown in Fig. 1G shows that αSMA levels in both the nucleus and cytosol increase around 15-20 fold during the

differentiation. Over the same timecourse, β-actin levels do not dramatically change, and nuclear accumulation is lower than cytosolic (n = 4 independent experiments using 2 different

control cell lines). All data are presented as mean ± standard deviation. Source data EXTENDED DATA FIG. 2 FORCED NUCLEAR LOCALIZATION OF ACTINS DOES NOT AFFECT MKL1 LEVELS OR LOCALIZATION.

A,B) Immunostaining of NLS-infected SMCs with Mkl1 antibody (A) shows no change in Mkl1 localization or intensity with α-NLS or β-NLS infection, quantified in (B). n = 14 cells infected with

empty vector (EV), 15 cells infected with β-NLS, and 11 cells infected with α-NLS across three independent staining experiments. Significance was assessed by one-way ANOVA with Tukey’s

multiple comparisons test. All data are presented as mean ± standard deviation. Source data EXTENDED DATA FIG. 3 MOUSE _ACTA2_SMC-R179C/+ SMOOTH MUSCLE CELLS HAVE DECREASED NUCLEAR ΑSMA AND

ARE HYPODIFFERENTIATED. A) Quantitation of immunoblot in Fig. 4E shows significant and reproducible decreases in accumulation of contractile proteins in the _Acta2_SMC-R179C/+ cells compared

with WT (n = 4 independent experiments). Significance was assessed by two-way ANOVA followed by Tukey’s multiple comparisons test. B) Quantitation of immunoblot in Fig. 4F shows decreased

nuclear accumulation, of αSMA in the _Acta2_SMC-R179C/+ cells compared with WT. β-actin accumulation to the nucleus was variable across experiments (n = 4 independent experiments).

Significance was assessed by two-way ANOVA followed by Tukey’s multiple comparisons test. C) Quantitation of immunoblot in Fig. 4G shows that LtA treatment further does moderately decrease

nuclear accumulation of β-actin in _Acta2_SMC-R179C/+ cells but does not significantly affect αSMA nuclear accumulation (n = 3 independent experiments). Significance was assessed by two-way

ANOVA followed by Tukey’s multiple comparisons test. D) Co-immunoprecipitation with Brg1 antibody confirms decreased association of both αSMA and β-actin with the Baf chromatin remodeling

complex in in _Acta2_SMC-R179C/+ SMCs which is partially rescued by TGFβ1 treatment (n = 3 independent experiments). All data are presented as mean ± standard deviation. Source data EXTENDED

DATA FIG. 4 DIFFERENTIAL CHROMATIN ACCESSIBILITY ALIGNS WITH TRANSCRIPTIONAL CHANGES IN _ACTA2_SMC-R179C/+ SMCS. A) Distribution of chromatin accessibility peaks around the transcription

start sites of nearby genes for peaks with increased accessibility in WT SMCs. B) Distribution of chromatin accessibility peaks around the transcription start sites of nearby genes for peaks

with increased accessibility in _Acta2_SMC-R179C/+ SMCs. C) Visualization of integrated scRNA-seq and ATAC-seq datasets plots the log2FC in gene expression _in vivo_ on the y-axis and the

log2FC in chromatin accessibility _in vitro_ on the x-axis. Genes indicated by dots inside the red circle have decreased accessibility and decreased expression in _Acta2_SMC-R179C/+ SMCs and

were analyzed by GO enrichment analysis in Fig. 5H. Genes indicated by dots inside the blue circle have increased accessibility and increased expression in _Acta2_SMC-R179C/+ SMCs and were

analyzed by GO enrichment analysis in Fig. 5H. EXTENDED DATA FIG. 5 GENES WITH INCREASED EXPRESSION AND ACCESSIBILITY IN _ACTA2_SMC-R179C/+ SMCS. A) Genome browser view for the _Klf4_ gene

shows chromatin accessibility increased in _Acta2_SMC-R179C/+ SMCs _in vitro_ and transcript levels visualized in UMAP space and quantified shows increased expression in _Acta2_SMC-R179C/+

SMCs _in vivo_. B) Genome browser view for the _Smad7_ gene shows chromatin accessibility increased in _Acta2_SMC-R179C/+ SMCs _in vitro_ and transcript levels visualized in UMAP space and

quantified shows increased expression in _Acta2_SMC-R179C/+ SMCs _in vivo_. P values denote results of Wilcoxon rank sum test with Bonferroni correction for multiple comparisons between all

‘SMC 1’ (5,816 cells) and ‘SMC 2’ (3,229 cells) in the scRNAseq dataset. For all violin plots, line depicts median, hinges depict interquartile range, whiskers depict 1.5 interquartile

ranges above and below the hinges. C) Quantitative RT-PCR validates shRNA-mediated knockdown of _Smad7_ in WT SMCs and shows knockdown of _Smad7_ increases expression of SMC contractile

genes. Graph shows three technical replicates, representative of three independent experiments from three distinct lentiviral infections. Significance was assessed by unpaired, two-tailed

t-test for each gene. All data are presented as mean ± standard deviation. Source data EXTENDED DATA FIG. 6 GENES WITH DECREASED EXPRESSION AND ACCESSIBILITY IN _ACTA2_SMC-R179C/+ SMCS. A)

Genome browser view for the _Itga1_ gene shows chromatin accessibility decreased in _Acta2_SMC-R179C/+ SMCs _in vitro_ and transcript levels visualized in UMAP space and quantified shows

decreased expression in _Acta2_SMC-R179C/+ SMCs _in vivo_. B) Genome browser view for the _Synpo2_ gene shows chromatin accessibility decreased in _Acta2_SMC-R179C/+ SMCs _in vitro_ and

transcript levels visualized in UMAP space and quantified shows decreased expression in _Acta2_SMC-R179C/+ SMCs _in vivo_. P values denote results of Wilcoxon rank sum test with Bonferroni

correction for multiple comparisons between all ‘SMC 1’ (5,816 cells) and ‘SMC 2’ (3,229 cells) in the scRNAseq dataset. For all violin plots, line depicts median, hinges depict

interquartile range, whiskers depict 1.5 interquartile ranges above and below the hinges. EXTENDED DATA FIG. 7 PARTICIPANT-DERIVED _ACTA2_ P.ARG179 SMCS ARE LESS DIFFERENTIATED THAN

CONTROLS. A) Demographic and genotype information for patient and control lines used in this study. B) Quantitation of immunoblot from Fig. 6A reveals significant decreases in contractile

protein accumulation in _ACTA2_ p.R179C patient SMCs compared with control SMCs (n = 4 independent experiments). Significance was assessed by one-way ANOVA with Sidak’s multiple comparisons

test. C-E) Immunoblot showing decreased contractile protein accumulation is a consistent finding across three _ACTA2_ p.R179 patient lines (C), quantified in (D,E). Graph shows biological

replicates- each dot represents one patient or control line. Significance in (D) ( + /- TGFβ1) was assessed by two-way ANOVA with Tukey’s multiple comparisons test. Significance in (E) (no

TGFβ1) was assessed by unpaired, two-tailed t-tests per protein. Grey bars/black dots represent control and green bars/dots represent _ACTA2_ p.R179. All data are presented as mean ± 

standard deviation. Source data EXTENDED DATA FIG. 8 PARTICIPANT-DERIVED _ACTA2_ P.ARG179 SMCS HAVE DECREASED NUCLEAR ΑSMA COMPARED WITH CONTROLS. A) Quantitation of immunoblot from Fig. 6C

shows a significant decrease in αSMA nuclear accumulation in patient-derived _ACTA2_ p.R179C SMCs compared with controls. β-actin accumulation to the nucleus was variable and not

significantly altered (n = 3 independent experiments). Significance was assessed by repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test. B,C) Immunoblot showing

decreased nuclear accumulation of αSMA is a consistent phenotype across three _ACTA2_ p.R179 patient lines (B), quantified in (C). Graph shows biological replicates- each dot represents one

patient or control line. Significance was assessed by unpaired, two-tailed t-test for each condition (cytosol, nucleus) for each protein. D) Quantitation of immunoblot from Fig. 6D shows a

significant increase in cytosolic αSMA and decrease in αSMA nuclear accumulation in patient-derived _ACTA2_ p.R179 NEPCs compared with controls. β-actin accumulation to the nucleus was not

significantly altered (n = 3 independent experiments). Significance was assessed by unpaired, two-tailed t-test for each condition (cytosol, nucleus) for each protein. E)

Co-immunoprecipitation with BRG1 antibody confirms decreased association of both αSMA and β-actin with the BAF chromatin remodeling complex in _ACTA2_ p.R179C SMCs. Grey bars/black dots

represent control and green bars/dots represent _ACTA2_ p.R179. All data are presented as mean ± standard deviation. Source data EXTENDED DATA FIG. 9 EXPRESSION OF SKELETAL Α-ACTIN CAN

COMPENSATE FOR LOSS OF NUCLEAR ΑSMA IN _ACTA2_-/- CELLS. A) Immunostaining for αSMA shows partial disruption of αSMA filaments in SMCs with the _ACTA2_ p.R179C pathogenic variant. B)

Subcellular fractionation immunoblot of _Acta2_ KO mouse SMCs shows that sarcomeric actin (encoded by _ACTA1_ and known to be expressed in _Acta2_ KO SMCs as a compensatory mechanism for

loss of αSMA) in these cells can go into the nucleus. C) Quantitative RT-PCR confirms that _ACTA2_ Crispr-edited iPSC-derived SMCs do not have increased expression of _ACTA1_. Graph shows

three technical replicates, results are representative of three independent experiments. All data are presented as mean ± standard deviation. Source data EXTENDED DATA FIG. 10 INTEGRATED

_ACTA2_ P.R179H AND MARFAN SYNDROME (MFS) AORTIC SCRNASEQ DATA. A) Complete dataset following reciprocal PCA data integration demonstrating representation of all major cell clusters in both

datasets. Red arrow indicates SMC cluster selected for downstream analysis. B) Expression plot of _MYH11_ confirming SMC identity for selected cluster. C) Overlaid UMAP plot for _ACTA2_

p.R179H and MFS SMCs demonstrating overrepresented sub-populations in _ACTA2_ patient (black arrows). D) Expression of ‘typical’ SMC modulation markers (_FN1_, _COL1A1_) with gradient of

expression in both patients. E) Expression of representative altered SMC phenotypic trajectory markers in _ACTA2_ patient (see Fig. 7I) not activated in MFS SMCs. F) Activation of

TGFβ1-responsive gene _SERPINE1_ is not observed in ACTA2 p.R179H SMCs as compared to MFS. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary discussion, Tables 1 and 2, Figs.

1–7 and source data and western blots for supplementary figures. REPORTING SUMMARY SUPPLEMENTARY DATA 1 Numerical source data for supplementary figures. SUPPLEMENTARY DATA 2 Image source

data for supplementary figures. SOURCE DATA SOURCE DATA FIG. 1 Unprocessed western blots, statistical source data and original image files. SOURCE DATA FIG. 2 Unprocessed western blots,

statistical source data and original image files. SOURCE DATA FIG. 3 Unprocessed western blots and statistical source data. SOURCE DATA FIG. 4 Unprocessed western blots, statistical source

data and original image files. SOURCE DATA FIG. 6 Unprocessed western blots, statistical source data and original image files. SOURCE DATA EXTENDED DATA FIG./TABLE 1 Statistical source data.

SOURCE DATA EXTENDED DATA FIG./TABLE 2 Statistical source data and original image files. SOURCE DATA EXTENDED DATA FIG./TABLE 3 Unprocessed western blots and statistical source data. SOURCE

DATA EXTENDED DATA FIG./TABLE 5 Statistical source data. SOURCE DATA EXTENDED DATA FIG./TABLE 7 Unprocessed western blots and statistical source data. SOURCE DATA EXTENDED DATA FIG./TABLE 8

Unprocessed western blots and statistical source data. SOURCE DATA EXTENDED DATA FIG./TABLE 9 Unprocessed western blots and statistical source data. RIGHTS AND PERMISSIONS Springer Nature

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Kwartler, C.S., Pedroza, A.J., Kaw, A. _et al._ Nuclear smooth muscle α-actin participates in vascular smooth muscle cell differentiation. _Nat Cardiovasc Res_ 2, 937–955 (2023).

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