Embryologic Origin Influences Smooth Muscle Cell Phenotypic Modulation Signatures in Murine Marfan Syndrome Aortic Aneurysm

Abstract

Background: Aortic root smooth muscle cells (SMC) develop from both the second heart field (SHF) and neural crest. Disparate responses to disease-causing Fbn1 variants by these lineages are proposed to promote focal aortic root aneurysm formation in Marfan syndrome (MFS), but lineage-stratified SMC analysis in vivo is lacking.

Methods: We generated SHF lineage-traced MFS mice and performed integrated multiomic (single-cell RNA and assay for transposase-accessible chromatin sequencing) analysis stratified by embryological origin. SMC subtypes were spatially identified via RNA in situ hybridization. Response to TWIST1 overexpression was determined via lentiviral transduction in human aortic SMCs.

Results: Lineage stratification enabled nuanced characterization of aortic root cells. We identified heightened SHF-derived SMC heterogeneity including a subset of Tnnt2 (cardiac troponin T)-expressing cells distinguished by altered proteoglycan expression. MFS aneurysm-associated SMC phenotypic modulation was identified in both SHF-traced and nontraced (neural crest-derived) SMCs; however, transcriptomic responses were distinct between lineages. SHF-derived modulated SMCs overexpressed collagen synthetic genes and small leucine-rich proteoglycans while nontraced SMCs activated chondrogenic genes. These modulated SMCs clustered focally in the aneurysmal aortic root at the region of SHF/neural crest lineage overlap. Integrated RNA-assay for transposase-accessible chromatin analysis identified enriched Twist1 and Smad2/3/4 complex binding motifs in SHF-derived modulated SMCs. TWIST1 overexpression promoted collagen and SLRP gene expression in vitro, suggesting TWIST1 may drive SHF-enriched collagen synthesis in MFS aneurysm.

Conclusions: SMCs derived from both SHF and neural crest lineages undergo phenotypic modulation in MFS aneurysm but are defined by subtly distinct transcriptional responses. Enhanced TWIST1 transcription factor activity may contribute to enriched collagen synthetic pathways SHF-derived SMCs in MFS.

Keywords: Marfan syndrome; aneurysm; extracellular matrix; neural crest; phenotype.

Figure 1:

Second heart field (SHF) lineage-tracing Marfan mouse development and scRNAseq workflow. A SHF lineage-tracing strategy. IRES-Cre recombinase cassette inserted into the 3’ untranslated region of Nkx2-5 locus enables upstream stop signal deletion in tdTomato gene inserted into Rosa26 locus, leading to permanent fluorescence B Stitched serial long axis images and inset 20X magnification images of a representative 16-week-old healthy control male SHF-traced mouse thoracic aorta. DAPI = nuclear counterstain, Elastin = green autofluorescence, tdTomato = red fluorescent signal from SHF-derived cells C Transthoracic echocardiography data for aortic root diameter in 16-week-old Fbn1^C1041G/+ (MFS) mice and littermate controls (WT). Line indicates mean diameter for n=6 animals of each sex. D Experimental workflow. Aortic tissue from the aortic valve to mid ascending aorta (dashed lines) digested to single cell suspension and sorted based on tdTomato fluorescent signal. Individual single cell (RNA) and nuclei (ATAC) libraries were generated and processed in parallel.

Figure 2:

Complete aortic root scRNAseq dataset. A Unsupervised clustering of >39,000 aortic root cells from MFS and control mice. EC = endothelial cell B Overlaid UMAP distributions of second heart field (SHF) and non-traced cell populations C Cell cluster breakdown by lineage. Each lineage comprises aggregated separate male and female samples (n=3 each sex) of both Fbn1^C1041G/+ and control genotype D Raw tdTomato mRNA transcript count per cell stratified by sorted lineage E Total scRNAseq dataset stratified by genotype demonstrates increased macrophage density (black arrow) and disease-associated smooth muscle cell cluster (red arrow) in MFS mice.

Figure 3:

Aortic SMC subcluster analysis. A UMAP projection of four distinct SMC subtypes B Enriched markers for each subcluster. Major SMC group (purple) is characterized by peak expression of mature contractile markers such as Myh11 compared to other subsets C SMC population distribution in UMAP space split into original sample subtypes (top) and pie charts for fraction of each SMC cluster in sample (bottom). Tnnt2+ SMCs (light blue) are enriched in SHF lineage regardless of genotype, while modSMCs (green) are largely limited to MFS samples. D Long axis chromogenic RNA in situ hybridization for Tnnt2 localizes this SMC cluster within the outer layers of the aortic root segment. E Heatmap for top up- and down-regulated genes in Tnnt2+ cluster compared to major SMC subset. F Violin plots for markers of interest in Tnnt2+ SMCs. Boxes denote interquartile range (IQR), line denotes median, whiskers extend 1.5 IQR from box hinges. p value <0.001 for all comparisons, log2FC denotes Tnnt2+ subset over main SMC cluster.

Figure 4:

Modulated SMC localization and phenotype stratified by lineage. A Proportion of all SMCs belonging to modSMC cluster by sample. SHF=lineage traced second heart field-derived cells, Non=untraced cells. B Short axis chromogenic RNA in-situ hybridization for SHF lineage marker (tdTomato) and insulin-like growth factor binding protein-2 (Igfbp2), a specific marker for the modSMC subset. 16-week-old MFS animals show modSMCs from both SHF (double stain, red arrowheads) and non-traced (blue stain only, blue arrowheads) while control animals demonstrate rare Igfbp2-positive cells (black arrowheads). Left image is stitched image of entire root circumference with boxes marking inset 20X magnification images. C Long-axis RNA localization shows modSMCs are concentrated in the MFS aortic root segment regardless of lineage (SHF-derived = red arrowheads, non-traced = blue arrowheads) while control animals show trivial Igfbp2 expression D Myh11 and Acta2 expression levels are similar between MFS modSMCs from SHF and non-traced lineages E Multiple collagen isoforms and proteoglycans are enriched in SHF-derived modSMCs (top panel) while Acan, Sost, and Tnfrsf11b are enriched in non-traced modSMCs (bottom). Boxes denote interquartile range (IQR), line denotes median, whiskers extend 1.5 IQR from box hinges. ‘ns’ indicates fold change not calculated between lineages due to non-significant difference, otherwise p < 0.001 for all genes with log2FC displayed.

Figure 5:

Single-cell ATAC dataset. A Unsupervised clustering of total scATAC data comprising MFS and control samples from SHF and non-traced lineages. Dashed lines indicate cells sub-selected for analysis B Enriched DNA accessibility peaks within the Myh11 gene confirm SMC identity of sub-selected clusters C UMAP projection of MFS SMCs reveals distinct profiles for SHF (red) and non-traced (blue) SMCs D GREAT output depicting top enriched biological processes for genes with regulatory regions corresponding to SHF-derived SMC marker peaks from the scATAC dataset E Integrated label transfer from scRNA dataset (left) onto scATAC dataset confirms modSMC enrichment in MFS samples. F ChromVAR output depicting top transcription factor DNA binding motifs in genome-wide assessment of enriched peak regions for modSMC subset (top) and quiescent SMC (bottom) from MFS mice only, all results statistically significant (p < 1x10⁻⁵⁰).

Figure 6:

Integrated scRNA/scATAC reveals Twist1 as a marker of SHF-SMC modulation. A Transcription factor binding motif accessibility signal for SHF-enriched motifs with enhanced signal in modSMC cluster compared to quiescent subset in MFS SMC/modSMC clusters. ‘ns’ indicates fold change not calculated between lineages due to non-significant difference, otherwise p < 0.001 where log2FC displayed. B Scatter plot for SMAD2:SMAD3:SMAD4 and TWIST1 motif activity for individual cells within the scATAC dataset. C Twist1 mRNA expression for SMC/modSMCs from individual samples within scRNA dataset (white diamonds indicate males, circles indicate females). Cells with at least one Twist1 transcripts considered positive. D Aortic tissue lysate western blots for aneurysmal segment (root/ascending) and non-dilated region (descending) from n=3 male 16-week-old mice with band density quantification relative to loading control (tubulin).

Figure 7:

TWIST1 overexpression induces fibrillar collagen and small leucine-rich proteoglycan expression in aortic SMCs. A RT-qPCR data for SHF-enriched modSMC markers from MFS mouse model (n=4 paired technical replicate values in n=2 distinct MFS patient aortic root SMC lines) following TWIST1 silencing (shRNA) or overexpression (OE). All values plotted as fold change (FC) relative to scrambled shRNA control. Dashed lines indicate FC=1 B RT-qPCR data for chondrogenic markers of non-SHF traced modSMCs from MFS mouse model C Chromatic accessibility tracks for single MFS aortic SMC line following stable lentiviral transduction of denoted construct. Blue bars indicate peaks called by MACS2 for total sample pool, grey boxes indicate first exon for denoted genes.