Differential Expression of Members of SOX Family of Transcription Factors in Failing Human Hearts

Abstract

The Sry-related high-mobility-group box (SOX) gene family, with 20 known transcription factors in humans, plays an essential role during development and disease processes. Several SOX proteins (SOX4, 11, and 9) are required for normal heart morphogenesis. SOX9 was shown to contribute to cardiac fibrosis. However, differential expression of other SOXs and their roles in the failing human myocardium have not been explored. Here we used the whole-transcriptome sequencing (RNA-seq), gene co-expression, and meta-analysis to examine whether any SOX factors might play a role in the failing human myocardium. RNA-seq analysis was performed for cardiac tissue samples from heart failure (HF) patients due to dilated cardiomyopathy (DCM), or hypertrophic cardiomyopathy (HCM) and healthy donors (NF). The RNA levels of 20 SOX genes from RNA-seq data were extracted and compared to the three groups. Four SOX genes whose RNA levels were significantly upregulated in DCM or HCM compared to NF. However, only SOX4 and SOX8 proteins were markedly increased in the HF groups. A moderate to strong correlation was observed between the RNA level of SOX4/8 and fibrotic genes among each individual. Gene co-expression network analysis identified genes associated and respond similarly to perturbations with SOX4 in cardiac tissues. Using a meta-analysis combining epigenetics and genome-wide association data, we reported several genomic variants associated with HF phenotype linked to SOX4 or SOX8. In summary, our results implicate that SOX4 and SOX8 have a role in cardiomyopathy, leading to HF in humans. The molecular mechanism associated with them in HF warrants further investigation.

Introduction

Heart failure (HF) is a pandemic condition affecting at least 26 million people worldwide¹. When a heart is failing, it may undergo cardiac remodeling with changes in cardiac structures and functions, leading to either dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM). These pathological features are largely driven by transcriptome reprogramming in response to pathophysiological stimulations such as oxidative stress and inflammation ^{2, 3}, which are primarily regulated by transcription factors (TFs). Increasing the knowledge of the transcriptional control mechanism, together with ensuring wider drug screening and repurposing capabilities, can be beneficial for developing novel therapies for HF.

The SRY-related high-mobility-group box (SOX) gene family, with 20 known TFs in humans, plays essential roles during embryonic development and cell fate determination as well as in many disease processes and functions, such as immunity and inflammation ⁴. It is known that SOX4 and SOX11 (members of the SOXC group) are critical for cardiac outflow tract formation, whereas SOX9 (a member of the SOXE group) is expressed in the cardiac cushion mesenchyme and is required for heart valve development ⁵. Additionally, SOX6, a member of the SOXD group, has been shown to regulate cardiac myocyte development ⁶. Genome-wide gene expression analysis of the infarcted mouse heart has revealed that SOX9 is a potential transcriptional regulator of the genes that mediate cardiac fibrosis ⁷. A recent study demonstrated that SOX9 regulates myocardial fibrosis during ischemic injury in animal models ⁸. SOX17 has been identified as a candidate risk gene for atrial hypertension with congenital heart disease ^{9, 10}. We previously identified a SOX transcription factor, SOX4, whose mRNA and protein levels were increased in the LV tissues from DCM samples ¹¹. However, whether the upregulation of SOX4 is present in the different types of cardiomyopathy is unknown. Additionally, differential post-natal expressions of other SOX TFs and their functional roles in the failing human myocardium have not been explored. Herein, we analyzed the whole-transcriptome data generated from Myocardial Applied Genomics Network (MAGNet) using left ventricle tissues of the hearts from DCM and HCM and from non-failing individuals (NF) to evaluate the expression of SOX genes and validate them in the human myocardium. Meta-analysis and molecular assays were carried out for two SOX genes, SOX4 and SOX 8, to further assess their potential roles in HF.

Methods

Human subject research

The research was carried out according to The Code of Ethics of the World Medical Association (Declaration of Helsinki), that informed consent was obtained. The study was approved by the Cleveland Clinic Institutional Review Board.

RNA-seq analysis.

RNA-seq data were extracted from data generated from the Myocardial Applied Genomics Network (MAGNet) (GSE141910) ¹², and data analyses were performed as previously described ¹³. Briefly, pair-end reads were mapped to the human hg 19 reference genome and then for transcript assembling and quantification using STAR package (v2.5.2b) with default parameters ¹⁴. Reads uniquely mapped were considered for further analysis. The count values were normalized to Transcripts Per Kilobase Million (TPM). Fold-change and false discovery rate (FDR) for differentially expressed genes were calculated using the R/Bioconductor packages linear models for microarray data (limma) ¹⁵. Genes that had their TPM level ≥ 1 in at least one sample type were used for downstream analysis. Changes ≥1.5-fold with a false discovery rate (FDR) adjusted p<0.05 were considered differentially expressed between groups. The online NetworkAnalyst ¹⁶ was used for co-expressed gene network analysis. Gene ontology analysis was carried out using Ingenuity pathway analysis (IPA, QIAGEN Inc., https://www.qiagenbioinformatics.com/). Gene correlation analysis was carried out using the correlation test in GraphPad Prism 9.

Western blot.

Protein extracts were prepared from human left ventricles (LV) from the same cohorts of subjects in MAGNet as previously described ¹³. Briefly, 60 mg of LV were homogenized using a tissue homogenizer with a 5×75mm Flat Bottom Stainless Steel generator probe (OMNI international, THP115) in a RIPA buffer (ThermoFisher, 89900) containing protease and phosphatase inhibitors (ThermoFisher, 78410 & 78420). Thirty µg total proteins were separated in SDS-polyacrylamide gel electrophoresis, transfer to nitrocellulose membranes or Polyvinylidene fluoride or polyvinylidene difluoride (PVDF), and blotted with primary antibodies overnight at 4°C. The total lane density of transferred proteins stained with Ponceau S was used to control for loading/transfer differences. Primary antibodies used were as follows: SOX4 (Diagenode, CS129100), SOX8 (GeneTex, GTX129949), SOX9 (Millipore, AB5535), SOX15 (ThermoFisher, 25415–1-AP). Secondary antibodies coupled to Alexa Fluor 680 (Invitrogen Molecular Probes) or IRDye 800 (LI-COR Biosciences) were used, and the Odyssey CLS infrared imager system (LI-COR Biosciences) was used for visualization of Western blot signals. Odyssey version 1.2 imaging software was used to process all images.

Cell culture Transfection, Real-Time Quantitative Reverse Transcription PCR

Mouse myogenic C2C12 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 12.5% fetal calf serum. The plasmids expressing the SOX4 or SOX8 proteins were made by Vectorbuilder Inc. (Chicago, US). Transfection was performed at 60% confluence with the indicated expression plasmid using ViaFect transfection reagent (Promega). Forty-eight hours after transfection, cells were collected for RNA purification. Total RNA of cells was prepared using RNeasy mini kit (Qiagen, 74104) followed manufacturer protocol. cDNA was synthesized using SuperScript™ IV First-Strand Synthesis System (Thermo Fisher Scientific, 18091050). qPCR was performed using a Quantstudio 3 Real-Time PCR system (Thermo Fisher Scientific). Primers and TaqMan® Gene Expression Assays are listed in Table S1. The Col1a1 and Lox gene expression experiments were performed in duplex real-time reactions. Each reaction was normalized to its endogenous control assay, e.g., VIC-labeled probe for 18S ribosomal RNA, to increase the precision and the fidelity of the measurement. Reactions were analyzed in triplicate, and relative mRNA levels were calculated using the 2^−ΔΔCT method and normalized to those of 18S ribosomal RNA. The statistical significance of fold changes among samples was calculated using GraphPad Prism 9 one-way ANOVA followed by Dunnett’s multiple comparison test ( p <0.05).

ChIP-seq data analysis

The SOX4 (GSE 104761) and SOX8 (GSE116344) ChIP-seq sequence files were downloaded from GEO. Sequence reads were mapped to the reference genome (hg 19) with Galaxy version Bowtie 2.4.2¹⁷ using default settings. The normalization and quantification of ChIP-seq reads and peak calling with MAC2 was performed as described previously¹³. Peak annotation was performed using HOMER software (4.9.1)¹⁸

Luciferase reporter assay

Candidate genomic regions were PCR-amplified using specific primers (Table S2 ), cloned into the pGL3 promoter-luciferase reporter plasmid (Promega, E1761 ). Cells were co-transfected with the appropriate reporter plasmid, a Renilla control plasmid and a combination of plasmids expressing SOX4 or SOX8 using ViaFect Transfection reagent (Promega, E4981). Cells were harvested 24 hr after transfection, and luciferase activity was determined using Firefly & Renilla Luciferase Assay Kit (Biotium, 30081) and the SpectraMax i3x (Molecular device) microplate reader. Reporter activities were normalized for transfection efficiency and calculated as means with standard deviation for technical triplicates. N = 3 biological replicates performed for each condition. Statistical significance was determined using Student’s t-test. P-value<0.05 was considered statistically significant.

Meta-analysis.

We analyzed the gene loci of interest using the Roadmap epigenomics ChromHMM 25-state model across all cardiac-tissue types and H3K27ac, H3K4me1 and H3K4me3 ChIPseq peak data in the left ventricle, vertebrate phastCons evolutionary conservation values ¹⁹ and genome-wide association study (GWAS) data from from the cardiovascular disease knowledge portal (CVDKP) ^{20, 21}. Only the genomic variants associated with the HF phenotype with a p-value < 0.05 and located within ± 50 kb of the gene of interest were analyzed.

Results

We carried out RNA-seq approach to investigate the whole-transcriptome profiles for left ventricle (LV) tissues from patients suffering from heart failure (HF) due to DCM (n = 162) and HCM (n = 28) and non-failing heart donors (n=162) as previously described ¹³. We identified 10,105 genes whose expression level is above 1 TPM (Transcripts Per Kilobase Million) in at least one of the three groups (Table S2). Among these 10,105 genes, there are 1,094 and 1473 genes whose RNA expression level differentially changed at least 1.5 fold in DCM or HCM, respectively ( Figure S1 and Table S3 –S5). Genes associated with HF, such as genes encoding the atrial natriuretic peptide, NPPA, (Fold change [FC] increased +6.3 in DCM/NF and +5.6 folds in HCM/NF ), B-type natriuretic peptide, NPPB, (FC +2.0 in DCM/NF and +1.8 in HCM/NF) and connective tissue growth factor, CTGF, (FC +1.8 in DCM/NF and HCM/NF) were robustly upregulated in DCM and HCM as expected (Figure S1). In contrast, the genes known to be down-regulated in HF, such as the gene that encods Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, ATP2A2, (FC decreased −1.9 in DCM/NF and −2.0 in HCM/NF), and α isoform myosin heavy chain, MYH6, (FC −6.8 in DCM/NF and −7.8 in HCM/NF) were significantly downregulated (Figure S1). Previously using gene ontology analysis, we found that DCM and HCM may share the same regulatory mechanisms in HF because more than half of the DEG genes overlapped between the two groups. The majority of the GO terms were commonly found in both groups (Figure S1 and Table S6-S9).

Previously we observed that the expression level of SOX4, one of the SOX transcription factor family members, was robustly increased in DCM LVs. To learn if the elevation of the SOX4 level was also increased in HCM and the expression level of the other SOX gene family members in heart failure, the transcriptome profiles of the SOX genes were extracted for comparisons.

Among 20 SOX genes, eight were expressed in at least one of the three groups (cut-off ≥ 1 TPM) (Table 1). The expressed SOX genes shared between the NF, DCM, and HCM groups are SOX4, SOX9, SOX7, SOX12, SOX17, and SOX18. Interestingly, these expressed SOX genes fall into three subgroups of SOX TF. They are SOXC, SOXE, and SOXF. All the SOXF group genes, including SOX7, SOX17, and SOX18, were present in NF, DCM, and HCM. Two of three SOXC genes, SOX4 and SOX12, were expressed in NF and HF. One SOXE gene, SOX9, was detected in all three groups. Two genes were expressed in failing human LVs but not in normal ones. They are SOX8, which belongs to the E group, and SOX15, a G group SOX gene. Some of these expressed SOX genes displayed differential expression between the non-failing and failing groups (fold changes ≥ 1.5). These included SOX4 (SOXC), SOX8, and SOX9 (SOXE), and SOX15 (SOXG). SOX4 RNA level was upregulated by 1.6- and 2.1-fold in DCM and HCM, respectively, compared to NF. In contrast, the SOX8 RNA level was significantly increased in DCM by 2.6-fold and in HCM by 2.8-fold compared to the NF LVs (Figure 1A & D and Table 1). To a lesser extent, SOX9, another E group gene, was increased by more than 1.5-fold in HCM but not in DCM (fold change 1.14; Figure 1G and Table 1). Additionally, SOX15, a solo member of SOXG, was increased by 1.9-fold in both DCM and HCM compared to NF. (Figure 1J and Table1). In contrast, other members of the SOX genes found to be expressed in the heart (SOX12, SOX7, SOX17, and SOX18) showed no significant differences between the NF and HF groups (Table 1).

Table 1.

List of all SOX genes RNA level in the human left ventricle

Group	SOX	NF	DCM	HCM	Fold change [DCM/NF]	Fold change [HCM/NF]	FDR
A	Sry	0.009062 ± 0.003347	0.00295 ± 0.004618	0.00295 ± 0.002950	n.d	n.d	n.d
B1	SOX1	0.001116 ± 0.0004903	0.003521 ± 0.001127	0.005152 ± 0.001921	n.d	n.d
	SOX2	0.3361 ± 0.02409	0.2474 ± 0.01995	0.2612 ± 0.05040	n.d	n.d	0.144204103
	SOX3	0.006036 ± 0.001629	0.006856 ± 0.001455	0.01279 ± 0.006416	n.d	n.d	n.d
B2	SOX14	n/a	n/a	n/a	n.d	n.d	n.d
	SOX21	0.003249 ± 8.050e-005	0.003263 ± 8.066e-005	0.00599 ± 0.003815	n.d	n.d	n.d
C	SOX4	3.107 ± 0.1512	5.08 ± 0.3159*	6.398 ± 0.7877*	1.63	2.06	0.000659261†
	SOX11	0.5313 ± 0.03375	0.6286 ± 0.03241	0.749 ± 0.09887	n.d	n.d	0.152700362
	SOX12	2.975 ± 0.1706	3.497 ± 0.1457	3.634 ± 0.3834	1.17	1.22	0.480099027
D	SOX5	0.01179 ± 0.0005469	0.01251 ± 0.001629	0.01149 ± 0.002472	n.d	n.d	n.d
	SOX6	0.04034 ± 0.001615	0.05744 ± 0.002764	0.06774 ± 0.007039	n.d	n.d	n.d
	SOX13	0.6468 ± 0.02202	0.6974 ± 0.02828	0.7598 ± 0.06953	1.07	1.17	0.68812168
E	SOX8	0.646 ± 0.04810	1.675 ± 0.1026*	1.815 ± 0.2003*	2.59	2.81	1.38E-08†
	SOX9	9.367 ± 0.4458	10.73 ± 0.5473	15.03 ± 1.449*	1.14	1.60	0.020963461†
	SOX10	0.2814 ± 0.01847	0.2616 ± 0.01441	0.2935 ± 0.03643	0.93	1.04	0.436887008
F	SOX7	4.469 ± 0.1750	5.183 ± 0.2518	6.208 ± 0.4956	1.16	1.39	0.214702805
	SOX17	8.93 ± 0.6237	10.77 ± 0.6649	10.72 ± 1.445	1.21	1.20	0.665101191
	SOX18	12.33 ± 1.105	13.13 ± 1.211	14.93 ± 3.284	1.06	1.21	0.602783863
G	SOX15	0.8379 ± 0.05817	1.584 ± 0.07436*	1.576 ± 0.1280*	1.89	1.88	1.02E-05†
H	SOX30	0.006429 ± 0.0008755	0.007274 ± 0.0008103	0.007515 ± 0.001856	n.d	n.d	n.d

Abbreviations: n/a, not available; n.d, not determined.

RNA level is present as Transcripts Per Kilobase Million (TPM).

^*P < 0.01 ANOVA.

^†False discovery rate (FDR) < 0.05.

Figure 1. Several SOX factors display differential expression between non-failing and end-stage heart failure human hearts.

(A, D, G, J) Showing mRNA level NF (n=162), DCM (n=162) and HCM (n=28) of SOX4 (A), SOX8 (D), SOX9 (G) and SOX15 (J) by RNA-seq. Data are shown as mean ± standard errors . Asterisks, p<0.0001 (Kruskal-Wallis test). (B,E, H, K) Showing protein level of SOX4 (B), SOX8 (E), SOX9 (H) and SOX15 (K) determined by Western blot and Ponceaus S staining (for actin) of the blot used for normalization. Immunoblot for GAPDH is presented as an additional reference. (C,F,I,L) Bar graphs showing the quantification of immunoblots for SOX4 (C), SOX8 (F), SOX9 (I) and SOX15 (L). The mean of biological replicates for each group with standard errors after normalized to Ponceaus was shown. The NF, non-failing heart, DCM, dilated cardiomyopathy, HCM, hypertrophic cardiomyopathy, Pon S, Ponceau S. Asterisk indicates p < 0.05 (Brown-Forsythe ANOVA test).

We validated the protein levels of the differentially expressed SOX genes found within the three groups using Western blot. The SOX4 protein level was significantly elevated more than 3-fold in DCM and HCM. (Figure 1B-C). The SOX8 protein in the NF group was barely detected, consistent with its RNA level (TPM < 1), whereas the SOX8 protein level increased robustly more than 5-fold in DCM and HCM (Figure 1E-F). However, the SOX9 and SOX15 protein levels were not significantly different among the three groups (Figure 1 H-I & K-L).

SOX4 and SOX8 act as transcriptional activators in various tissue and organ systems. In order to learn how SOX4 and SOX8 coordinated with other genes in the end-stage hearts, we performed a correlation analysis between SOX4 or SOX8 and the 1244 genes upregulated in either DCM or HCM for each individual. The correlation analysis revealed that more than 50% of upregulated genes had moderate to strong correlation strength with either SOX4 or SOX8 (Figure 2A-B). These genes included HF-associated genes such as NPPA (Natriuretic Peptide A) and fibrosis-associated genes such as CTGF (connective tissue growth factor)²², POSTN (Perostin), Col1a1 (collagen type I alpha 1) ²³ and a collagen cross-link enzyme, LOX (Lysyla oxidase) ²⁴ (Figure 2C).

Figure 2. SOX4 and SOX8 are linked with genes associated with heart failure (HF).

(A) Showing heat map of Person’s correlation analysis of each individual between the RNA level of the 1224 upregulated genes identified from end-stage heart failure (DCM and HCM) human hearts and SOX4 or SOX8. The ranking is based on the correlation coefficient between each gene and SOX4. (B) Summary of correlation strength of the RNA level of SOX4 or SOX8 and 1224 upregulated genes in end-stage heart failure (DCM and HCM) human hearts. (C) Person’s correlation analysis of the RNA level of SOX4 or SOX8 and other HF-associated genes. (D) Bar graphs showing the RNA levels in NF (n=162), DCM (n=162), HCM (n=28) from RNA-seq analysis for VIM, SNAL1, VCAM1 and FZD7. (E) Person’s correlation analysis of RNA level of SOX4 or SOX8 and VIM, SNAL1, VCAM1, and FZD7. (F) Relative fold changes in the levels of mRNA for SOX4 .and SOX8 Snail1, Ezh2, and Vcam1 determined by qRT-PCR in transfection assays. (G) Relative fold changes in the level of mRNA for Ctgf, Postn, Col1a1 , Lox. 18s mRNA levels were used for normalization. Data are the mean ± SD of triplicate cultures per condition. * p < 0.05, ** p< 0.01, *** p<0.0001. ns: not significant. (one-way ANOVA with Dunnett’s multiple comparison test). (H) Vocanol plots showing the genes associated with SOX4 peak (S4 gene, green dot ) and with SOX8 (S8 gene, blue dot) among the differentially expressed genes (DEG) in DCM and HCM. The genes with a p-value inferior to 0.05 (-log10 p-value superior to 1.3) and at least a 1.5 fold change (log10(FC) greater than 0.176 or less than −0.176 ) were considered as DEGs. The red dot represents upregulated DEGs. The black dot represents downregulated DEGs. The Gray dot represents non-significant genes UP: upregulation, DOWN: downregulation, Not sig: not significant. (I) Luciferase reporter activity of tested putative genomic regions for CTGF, COL1A1, and FZD7. Data are presented as means with a standard deviation of technical triplicates in an experiment representative of at least three independent experiments. Statistical significance was determined using Student’s t-test. p-value<0.05 was considered statistically significant.

Both SOX4 and SOX8 have been shown to control the expression of genes involved in epithelial-to-mesenchymal transition (EMT) and cell proliferation during the development of tissues/organs and cancers ²⁵. Interestingly, we also observed several genes involved in EMT or cell proliferation known for downstream gene targets of SOX4 or SOX8 that were upregulated significantly in end-stage hearts, such as VIM (Vimentin), SNAI1 (Snail family transcriptional repressor 1), VACM1 (vascular cell adhesion protein one and FZD7 (Frizzled-7) ^{26, 27} (Figure 2D). The correlation analysis revealed a strong or moderate correlation of these genes with either SOX4 or SOX8 (Figure 2E).

To further investigate the transcriptional effect of SOX4 and SOX8, we overexpressed each factor in an early myoblast C2C12 cell line and assayed gene expression changes by qRT-PCR. After 48 hours of transfection, the expression of SOX4 or SOX8 was robustly increased in the C2C12 cells (Figure 2F). As expected, SOX4 overexpression increased the expression levels of Ezh2, Snail1, Vcam1 and Fzd7 that are known downstream gene targets of SOX4 or SOX8. Interestingly, these genes were also reported to be involved in cardiac fibrosis ^{28, 29}. Vcam1 expression was also increased in SOX8 overexpressed C2C12 cells (Figure 2F). Most interestingly, overexpression of SOX4 or SOX8 in C2C12 cells resulted in upregulation of the expression level of fibrosis markers Ctgf, Col1a1, and Lox, (Figure 2G). The ectopic overexpression of SOX4 or SOX8 did not change the RNA level of Postn (Figure 2G).

To further understand the involvement of SOX4 and SOX8 transcriptional activity in pathological processes in end-stage human hearts, we compared the differential expressed genes (DEG) identified from our RNA-seq analysis to the SOX4 and SOX8 known downstream gene targets identified from the published SOX4 and SOX8 ChIP-seq data^{27, 30, 31}. There were 298 (27% of DEGs) genes associated with SOX4 peaks and 283 (26% of DEGs) genes associated with SOX8 peaks among the DEGs in the DCM group, whereas 356 (24%) and 333 (23%) genes were associated with SOX4 and SOX8 peak respectively in HCM groups (Figure 2H, Table S10-13). These data suggest that SOX4 and SOX8 are profoundly involved in regulating gene expression in heart failure. Additionally, we generated luciferase reporter constructs containing the putative SOX4 or SOX8 regulatory regions based on the published data for CTGF, LOX, COL1A1, and FZD7 ^{27, 30}. Based on JASPER analysis, the selected genomic regions contained several SOX4 and SOX8 DNA binding motifs (Figure S2). SOX4 alone induced the luciferase expression in FZD (−1.4 kb) upstream 1.4 kb, CTGF(−3.2 kb), CTGF (−45 kb) significantly but not in and LOX (−1 kb & + 0.1kb) compared to the non-SOX protein expressed group whereas SOX8 alone induced reporter activity in CTGF (−3.2 kb) (Figrue 2 I). Co-expression of SOX4 and SOX8 resulted in significant upregulation of luciferase activity in FZD (−1.4 kb) CTGF (−3.2 kb), and CTGF (−45 kb) regions compare to either SOX4 or SOX8 alone (Figure 2 I). To a lesser extent, the COL1A1 (+ 1 kb) region was increased in the presence of SOX4 but not SOX8, and the co-expression of both SOX proteins upregulated the luciferase activity COL1A1 (−8.4kb region) (Figure 2I). Together, these data suggest that SOX4 and SOX8 may be involved in the fibrosis process by directly controlling the expression of fibrotic genes.

The gene network analysis of genes expressed in HCM or DCM using co-expressed gene network analysis showed several genes associated with SOX4 in the left ventricle of hearts. These genes included collagen-related genes, COL6A1 and COL6A2, a putative calcium-binding protein, reticulocalbin-3 (RCN3), an enzyme involved in cell migration proliferation and the epithelial-to-mesenchymal transition, dihydropyrimidinase-related protein 3 (DPYSL3) ³², a secreted sulfated glycoprotein, C-type lectin domain containing 11A (CLEC11A), an actin-binding protein, retinoic acid-induced protein 14 (RAI14), and an ETS transcription factor ELK3 (ELK3) (Figure 3A). However, we did not observe any significant genes linked to SOX8. Among the SOX4-associated genes, CLEC11A, COL6A2, DPYSL3, and RCN3 were upregulated significantly at least 1.5-fold in both DCM and HCM, whereas COL6A1 and ELK3 were only found to have increased considerably in DCM or HCM (Figure 3B).

Figure 3. Co-expressed gene network analysis reveals SOX4-associated genes upregulated in failing human myocardium.

(A) Showing network Co-expressed analysis for SOX4 (B) Showing mRNA level NF. (n=162), DCM (n=162) and HCM (n=28) of CLEC11A, COL6A1, COL6A2, ELK3, DPYSL3 and RCN3 by RNA-seq. Data are shown as mean± SE Asterisks, p<0.0001 (Kruskal-Wallis test). ns, not significant.

We subsequently analyzed chromatin state data and integrated them with the GWAS signals for both SOX4 and SOX8 loci from public data resources. We included all the GWAS signals and variants associated with the HF phenotype from the cardiovascular disease knowledge portal (CVDKP) ^{20, 21} and compared them with the Roadmap epigenomics data ¹⁹. Roadmap epigenomics revealed several active enhancers along with transcriptional activity at both gene loci in fetal heart, left and right ventricular, and atrial tissues (Figure 4). Several genomic variants with a p-value of < 0.05 were found within ± 20 kilobases (kb) of the SOX4 and SOX8 loci and overlapped with active cis-regulatory element signatures in the fetal heart and left and right ventricular tissues. At the SOX4 loci, one variant, rs192898967, located upstream 5.8 kb, overlapped with the DNase hypersensitivity signal, H3K27ac, the H3K4me1 enhancer signatures, and the H3K4me3 promoter mark, indicating the variant is associated with cis-regulatory elements, which may interact with a trans-acting element to regulate the transcription of SOX4. Additionally, variants 2–4 and 5–9 were located at 3’ UTR and downstream of the gene, respectively (Figure 4A). The frequency of the variants 5–9 was more common than others. At the SOX8 loci, a wide variety of cis-regulatory elements was present. However, its neighboring gene, LMF1 (Lipase Maturation Factor 1), is only 10 kb away from the transcription start site of SOX8. Therefore, both genes may share the same cis-regulatory elements. Nevertheless, we observed two variants, rs12448761, and rs552159903, which were located 7kb and 12 kb upstream of SOX8 that also belonged to the intron of LMF1. These regions overlapped with the active enhancer marks as well as the DNase hypersensitivity peaks, suggesting they may be involved in controlling the expression of both genes (Figure 4B). There are eight additional variants found in the SOX8 coding regions, at 3’ UTR, and in the downstream regions. Even though these eight variants were not linked with active cis-regulatory elements, their occurrence was more frequent than that of the other two variants located upstream of SOX8. Therefore, these eight variants may be more likely to contribute to the gene expression level changes.

Figure 4. Epigenetic overview and significant genomic variants associated with heart failure phenotype in the SOX4 and SOX8 loci.

Roadmap CHromHMM 25-state model annotations in cardiac tissues, H3K27ac, H3K4me1, H3K4me3 peaks in the left ventricle, Vertebrate phastCons evolutionary conservations values and SNP from cardiovascular disease knowledge portal (CVDKP) in SOX4 (A) and SOX8 (B). The significant variants (p< 0.05) associated with cis-regulatory elements or located within the gene body and within ± 20 kb of SOX4 or SOX8 are listed.

Discussion

To our knowledge, the present study is the first to systematically analyze SOX family protein myocardial tissue levels in different types of cardiomyopathies leading to HF in humans. We identified four differentially expressed SOX genes between non-failing and failing human hearts. The protein levels of two genes, namely SOX4 and SOX8, were elevated in the left ventricle tissues of hearts from patients who had DCM and HCM compared to the NF controls.

We had previously reported that SOX4 protein and RNA levels were significantly elevated in DCM tissues ¹³. Here, we further analyzed its protein and RNA levels in HCM tissues to confirm these findings (Figure 1). Our data suggest that the elevation of SOX4 is associated with a shared common pathway present in the development and progression of HF. However, the expression levels of SOX4 in other cardiovascular conditions causing HF remains to be determined. Correlation analysis showed a moderate to strong correlation between the RNA level of SOX4 or SOX8 and genes upregulated in HF groups, including genes that were shown to be involved in cardiac fibrosis, suggesting a transcriptional regulation of these genes by SOX4 or SOX8 (Figure 2A-C). SOX4 was previously shown to bind to the genomic regions associated with pro-angiogenic process ³⁰, which is one of the contributors to HF and cardiac fibrosis. Using reporter assay, we showed that SOX4 or SOX8 might directly regulate the expression of fibrotic genes, e.g., CTGF and COL1A1 (Figure 2I). Although co-expression of SOX4 or SOX8 did not increase reporter activity in the two tested LOX genomic regions, this did not exclude that SOX4 or SOX8 may directly control LOX gene expression by binding to other SOX-bound enhancers of the gene in the human heart. It would be important to establish whole-genomic occupancy for SOX4 and SOX8 in human hearts, allowing to further study the involvement of SOX4 and SOX8 in the pathological processes of failing hearts.

Using co-expressed gene-associated network analysis, we found that CLEC11A, COL6A2, DPYSL3, and RCN3 were positively associated with SOX4 (Figure 3). Interestingly, CLEC11A, COL6A2, RCN3, and SOX4 are all involved in the fibrosis of various tissues ^33–36. Therefore, it would be intriguing to investigate whether they may contribute to the development of cardiac fibrosis. Further, DPYSL3 has been shown to regulate cell mitosis, migration, and epithelial-mesenchymal transition processes in breast cancer ³². These cellular events are all associated with the pathological processes of HF. It will be interesting to further explore the role of these processes and their relationship with SOX4 in HF ELK3 was found to be significantly upregulated in HCM but not in DCM. The gene was known for its role in the inhibition of iNOS ³⁷. Interestingly, it has been shown that the mRNA of iNOS can be detected in endomyocardial tissues from patients with DCM but not HCM ³⁸. The upregulation of ELK3 in HCM suggests the involvement of this TF in suppressing the expression of iNOS in HCM. Nevertheless, the role of ELK3 in HF has not been reported.

Although we did not find any genes associated with SOX8 in the human LV, this does not exclude the involvement of SOX8 in regulating cardiac function, especially in earlier pathogenic processes. This is because most SOX8 studies have been focused on its role in reproduction, neural development, and cancers ^39–41. Overexpression of SOX8 in early myocytes upregulated the known cardiac fibrosis genes (Figure 2G), suggesting the involvement of SOX8 in cardiac fibrosis. Our correlation analysis further supported this notion as the RNA level of many fibrotic genes, such as CTGF, was positively correlated with the level of SOX8 (Figure 2C). In addition, SOX8 belongs to the same SOXE group as SOX9, which plays a key role in regulating cardiac fibrosis in HF secondary to ischemic heart disease ⁷. The ablation of Sox9 in cardiomyocytes only delayed cardiac hypertrophy and fibrosis after cardiac pressure overload ⁴², suggesting other mechanisms contribute to the cardiac fibrosis in HF. Because a functional redundancy occurs in the same family of SOX factors, SOX8 may elicit its function similarly to SOX9 in cardiac fibrosis. Interestingly, we did not detect a significant change of the SOX9 protein in NF, DCM, and HCM despite the fact that its RNA level was increased in DCM and HCM (Figure 1 G-I). It has been reported that SOX9-expressing cells were found near the infarcted area in human ischemic hearts ⁷. Therefore, SOX9-positive cells may only be found regionally close to the fibrotic regions of LVs in DCM and HCM. Furthermore, unlike SOX9, both SOX8 RNA and protein levels were significantly elevated in DCM and HCM (Figure 1 D-F), suggesting SOX8 operates differently in regulating the pathological processes of the heart. Several SOX transcription factors, such as SOX4 and SOX9, have been linked to inflammation^{8, 43}, which promotes cardiac fibrosis and is a crucial factor in the pathogenesis and progression of HF ⁴⁴. Therefore SOX4 and SOX8 may have a role in the pathogenesis and progression of the DCM and HCM, leading to HF. It will be interesting to explore this possibility further.

Using a meta-analysis combining Roadmap Epigenomics and GWAS data from CVDKP for SOX4 and SOX8 loci, we reported several genomic variants associated with HF (Figure 4). Some of these variants are within the DNA regions related to active cis-regulatory elements, such as enhancers, suggesting their potential involvement in gene regulation. However, further functional validation of these variants will be necessary to determine whether they contribute to the gene expression level of SOX4 and SOX8 and whether they are risk alleles for HF.

Gene expression level is precisely controlled by TFs and the transcriptional machinery associated with them. Thus, targeting TFs to manipulate gene activation or inhibition, specifically in the desired conditions, is an innovative approach for therapeutic purposes. As TFs, SOX proteins elicit their transcriptional function by recognizing and binding to a specific DNA sequence motif and recruiting their specific TF working partners, such as SOX9 and steroidogenic factor 1 in gonadal development; or by dimerizing themselves, such as by SOX9 dimerization in chondrogenesis. Therefore, it is logical to target SOX TFs by interrupting their protein-protein interactions or by interfering with their binding to the specific DNA sequencing ⁴⁵. However, at present, researchers have only been able to design an efficient method to target one SOX factor, SOX18, to mitigate the progression of cancer ⁴⁶. This is mainly because (1) the same family of SOX proteins usually operates in a mutually redundant manner, (2) SOX proteins often have multiple protein partners, and (3) the delivery of the drug to the nucleus for targeting TFs is challenging. Therefore, understanding the protein structure, the organization of the protein-protein interaction, and the protein-DNA interface for each SOX protein at the molecular level will allow logical designs of treatment to be effective, especially aiming at these features. Additionally, the protein-binding partners of SOX4 and SOX8 remain unknown. Thus, it is essential to study the protein-protein interactions for both SOX proteins in the process of HF in the future.

The posttranslational regulation of the SOX protein has been shown to play a vital role in regulating SOX protein activity and stability. Acetylation of the SOX protein at specific residues can promote transcriptional activity, whereas phosphorylation or SUMOylation can cause the degradation of SOX proteins ^{47, 48}. For example, it was shown that the acetylation of SOX4 at the lysine 95 position resulted in chromatin remodeling during myoblast differentiation ⁴⁹. Targeting the enzymes responsible for these posttranslational modifications (PTM) will be another promising approach for treating HF drug discovery. Understanding the PTM’s machinery for SOX proteins in hearts, particularly for SOX4, SOX8, and their protein families SOXC and E, will facilitate drug discovery for HF.

In summary, we identified two differentially expressed SOX TFs, SOX4 and SOX8, and their associated genes in human left ventricle tissues between non-failing and failing hearts. Several genomic variants linked with SOX4 and SOX8 were reported in this study, and further elucidation of their functional roles and underlying molecular mechanisms is warranted.

Brief Commentary.

Background

Heart failure (HF) affects 5.7 million people in the U.S. with more than 500,000 new cases added annually. New therapeutic strategies remain needed for HF. The cardiac-pathological features are driven by transcriptome reprogramming. Understanding transcriptional control of HF is beneficial for developing novel therapies.

Translational Significance

This study revealed that SOX4 and SOX8 were significantly increased in cardiac tissues of HF patients and the HF-genetic variants and epigenetic changes associated with these two genes. Understanding the role of SOX4 and SOX8 in failing human myocardium and gene regulatory networks related to them may help to identify potential therapeutic targets for HF.

Abbreviations:

SOX

Sry-related high-mobility-group box

HF

Heart failure

NF

non-failing

DCM

Dilated cardiomyopathy

HCM

hypertrophic cardiomyopathy (HCM)

MAGNet

Myocardial Applied Genomics Network

TF

Transcirption factor

TPM

Transcripts Per Kilobase Million

GWAS

Genome-wide association study

RCN3

Reticulocalbin-3

DPYSL3

dihydropyrimidinase-related protein 3

CLEC11A

C-type lectin domain containing 11A

ELK3

ETS transcription factor 3

RAI14

Retinoic acid-induced protein 14

LMF1

Lipase Maturation Factor 1

PTM

Posttranslational modifications