Tomo-Seq Identifies SOX9 as a Key Regulator of Cardiac Fibrosis during Ischemic Injury

Background: Cardiac ischemic injury induces a pathological remodeling response, which can ultimately lead to heart failure. Detailed mechanistic insights into molecular signaling pathways relevant for different aspects of cardiac remodeling will support the identification of novel therapeutic targets. Methods: Although genome-wide transcriptome analysis on diseased tissues has greatly advanced our understanding of the regulatory networks that drive pathological changes in the heart, this approach has been disadvantaged by the fact that the signals are derived from tissue homogenates. Here we used tomo-seq to obtain a genome-wide gene expression signature with high spatial resolution spanning from the infarcted area to the remote to identify new regulators of cardiac remodeling. Cardiac tissue samples from patients suffering from ischemic heart disease were used to validate our findings. Results: Tracing transcriptional differences with a high spatial resolution across the infarcted heart enabled us to identify gene clusters that share a comparable expression profile. The spatial distribution patterns indicated a separation of expressional changes for genes involved in specific aspects of cardiac remodeling, such as fibrosis, cardiomyocyte hypertrophy, and calcium handling (Col1a2, Nppa, and Serca2). Subsequent correlation analysis allowed for the identification of novel factors that share a comparable transcriptional regulation pattern across the infarcted tissue. The strong correlation between the expression levels of these known marker genes and the expression of the coregulated genes could be confirmed in human ischemic cardiac tissue samples. Follow-up analysis identified SOX9 as common transcriptional regulator of a large portion of the fibrosis-related genes that become activated under conditions of ischemic injury. Lineage-tracing experiments indicated that the majority of COL1-positive fibroblasts stem from a pool of SOX9-expressing cells, and in vivo loss of Sox9 blunted the cardiac fibrotic response on ischemic injury. The colocalization between SOX9 and COL1 could also be confirmed in patients suffering from ischemic heart disease. Conclusions: Based on the exact local expression cues, tomo-seq can serve to reveal novel genes and key transcription factors involved in specific aspects of cardiac remodeling. Using tomo-seq, we were able to unveil the unknown relevance of SOX9 as a key regulator of cardiac fibrosis, pointing to SOX9 as a potential therapeutic target for cardiac fibrosis.