myocardial myocardial Eva van ◼ osis fibr SOX9 transcription ◼ PhD* PhD PhD PhD MSc, PhD naarden, MSc, PhD naarden, PhD* ventricular remodeling Correspondence to: Correspondence Rooij, MSc, PhD, Hubrecht Department of Institute, KNAW University Medical Cardiology, Uppsalalaan 8, Center Utrecht, The Netherlands. 3584CT Utrecht, E-mail [email protected] of Funding, see page 1408 Sources Key Words: ischemia ◼ © 2017 American Heart Association, Inc. *Drs. Lacraz and Junker contributed equally. Grégory P.A. Lacraz, MSc, P.A. Grégory MSc, Jan Philipp Junker, Monika M. Gladka, MSc, MSc Bas Molenaar, Scholman, MSc Koen T. MSc Marta Vigil-Garcia, BS Danielle Versteeg, BS Hesther de Ruiter, MSc, Vermunt, Marit W. MSc, Creyghton, Menno P. Manon M.H. Huibers, Nicolaas de Jonge, MD Alexander van Oude- Eva van Rooij, MSc, PhD 2017;136:1396–1409. DOI: 10.1161/CIRCULATIONAHA.117.027832 DOI: 2017;136:1396–1409. Circulation. blunted the cardiac fibrotic fibrotic blunted the cardiac Sox9 ). Subsequent correlation analysis allowed). Subsequent correlation Serca2 Editorial, see p 1410 , and Nppa Based on the exact local expression cues, tomo-seq can Based on the exact local expression Cardiac ischemic injury induces a pathological remodeling ischemic injury induces a pathological remodeling Cardiac , Although genome-wide transcriptome analysis on diseased tissues Tracing transcriptional differences with a high spatial resolution with a high spatial resolution transcriptional differences Tracing Col1a2 October 10, 2017 October 10, 1396 CONCLUSIONS: novel genes and key transcription factors involved in specific serve to reveal able to unveil the Using tomo-seq, we were remodeling. aspects of cardiac pointing fibrosis, of cardiac of SOX9 as a key regulator unknown relevance fibrosis. to SOX9 as a potential therapeutic target for cardiac for the identification of novel factors that share a comparable transcriptionalfor the identification of novel factors that share between correlation tissue. The strong pattern the infarcted regulation across of levels of these known marker genes and the expression the expression tissue genes could be confirmed in human ischemic cardiac the coregulated samples. Follow-up analysis identified SOX9 as common transcriptional genes that become of a large portion of the fibrosis-related regulator Lineage-tracing experiments activated under conditions of ischemic injury. a pool of stem from indicated that the majority of COL1-positive fibroblasts cells, and in vivo loss of SOX9-expressing The colocalization between SOX9 and COL1 on ischemic injury. response ischemic heart disease. from could also be confirmed in patients suffering RESULTS: a heart enabled us to identify gene clusters that share the infarcted across The spatial distribution patterns profile. indicated a comparable expression changes for genes involved in specific aspects ofseparation of expressional and calcium hypertrophy, cardiomyocyte such as fibrosis, remodeling, cardiac handling ( METHODS: networks that drive advanced our understanding of the regulatory has greatly has been disadvantaged bypathological changes in the heart, this approach we used tissue homogenates. Here derived from the fact that the signals are with high spatial signature tomo-seq to obtain a genome-wide gene expression to identify new to the remote area the infarcted spanning from resolution patients tissue samples from Cardiac remodeling. of cardiac regulators used to validate our findings. ischemic heart disease were from suffering BACKGROUND: Detailed mechanistic which can ultimately lead to heart failure. response, aspects of for different insights into molecular signaling pathways relevant will support the identification of novel therapeutic targets. remodeling cardiac Ischemic Injury Regulator of Cardiac Fibrosis During Fibrosis Regulator of Cardiac Tomo-Seq Identifies SOX9 as a Key Tomo-Seq ORIGINAL RESEARCH ARTICLE Downloaded from http://circ.ahajournals.org/ by guest on February 6, 2018 SOX9 as Key Regulator of Cardiac Fibrosis ORIGINAL RESEARCH transcriptional regulation pattern across the infarct- Clinical Perspective ed tissue. Subsequent functional annotation analy- ARTICLE sis indicated that these genes could be linked to the What Is New? known gene function of their reference gene. The strong correlation between the expression levels of • SOX9 is a key regulator of cardiac fibrosis after the marker genes and the expression of the coregu- ischemic injury in mice by regulating the expression of many extracellular matrix-related proteins. lated genes could be confirmed in human ischemic • SOX9 is induced in cardiac tissue from patients suf- tissue samples. fering from ischemic heart disease and colocalizes Our data show that the high spatial resolution in gene with COL1 expression. expression signatures obtained by tomo-seq reveals new • Reduced levels of SOX9 lead to less cardiac fibrosis regulators, genetic pathways, and transcription factors after ischemic injury in mice. that are active in well-defined regions of the heart and • Tomo-seq can be used to identify new players in potentially involved in specific aspects of heart disease. cardiac biology and disease. Using this technique, we identified SOX9 as a potent regulator of many of the Col1a2 coregulated genes. In What Are the Clinical Implications? vivo loss of Sox9 reduced the expression of many ex- • Our data suggest that therapeutic inhibition of tracellular matrix (ECM) genes, which coincided with Downloaded from SOX9 in the diseased heart could lead to a reduc- a blunted cardiac fibrotic response on ischemic injury. tion in cardiac fibrosis. These data unveil the currently unknown relevance of SOX9 as a key regulator of cardiac fibrosis and under- scores that tomo-seq can be used to increase our mech- schemic heart disease induces a heterogeneous re- anistic insights into cardiac remodeling to help guide the http://circ.ahajournals.org/ modeling response across the damaged area that in- identification of novel therapeutic candidates. Ivolves fibroblast activation, cardiomyocyte hypertro- phy, and changes in calcium handling, all of which are METHODS eventually detrimental for cardiac function.1,2 Fibroblast An expanded Methods section is available in the online-only activation and cardiomyocyte hypertrophy occur as a Data Supplement. All reported gene expression data are avail- direct effect of the local stress signals caused by the able in the online-only Data Supplement. loss of viable tissue in the infarcted area. Subsequently, by guest on February 6, 2018 a decline in contractility of the surviving cardiomyocytes occurs, caused by a change in metabolism and calcium- Ischemia Reperfusion Model handling genes.3 Animal experiments were performed in accordance with the Genome-wide transcriptome analysis on extracts institutional review committee at the Hubrecht Institute. Mice were randomly subjected to either sham or ischemia reperfu- from diseased tissues has significantly enhanced our sion surgery as previously described.7 Two weeks after sur- understanding of the gene regulatory networks that gery, cardiac tissues were collected for further analysis. drive these pathological changes in the heart.4,5 How- ever, to date, these approaches have been disadvan- taged by the fact that the signals are derived from Tomo-Seq tissue homogenates, which inherently causes the loss Tomo-seq experiments were performed as described else- where.6 In short, 2.5-mm-wide portions of cardiac mouse of spatial information and dilutes out more localized tissue spanning from the infarct toward the remote region expression signatures. Recent developments in RNA of the left ventricular anterior wall were embedded in tis- amplification strategies provide the opportunity to sue-freezing medium, frozen on dry ice, and cryosectioned use small amounts of input RNA for genome-wide into 48 slices of 80 μm thickness. We extracted RNA from sequencing. Here we use tomo-seq6 to obtain a ge- individual slices and prepared barcoded Illumina sequenc- nome-wide gene expression signature with high spa- ing libraries according to the CEL-seq protocol.8 Paired-end tial resolution spanning from the infarcted area to reads obtained by Illumina sequencing were aligned to the remote. Tracing transcriptional differences across the transcriptome using Burrows-Wheeler Aligner’s Smith- the infarcted heart enabled us to identify clusters of Waterman alignment.9 The 5’ mate of each pair was used genes with a comparable gene expression profile. In for mapping, discarding all reads that mapped equally well these individual clusters, we recognized genes with to multiple loci. The 3’ mate was used for barcode informa- tion. Reads counts were normalized to the same number well-known functions in specific aspects of heart re- of total reads per section. Tomo-seq data analysis was per- modeling, such as Col1a2 for fibrosis, Nppa for car- formed in MATLAB (MathWorks) using custom-written code. diomyocyte hypertrophy, or Serca2 for contractility. For data analysis, we used an expression cutoff of >4 reads Correlation analyses using the spatial distribution in >1 section. In differential expression analysis (Figure 1C), patterns of these marker genes allowed for the iden- we determined the boundary between remote and infarcted tification of novel factors that share a comparable zone based on the spatial partitioning detected by pairwise Circulation. 2017;136:1396–1409. DOI: 10.1161/CIRCULATIONAHA.117.027832 October 10, 2017 1397 Lacraz et al Whole genomeDiff.