Spatial transcriptomics unveils ZBTB11 as a regulator of cardiomyocyte degeneration in arrhythmogenic cardiomyopathy.

AIMS: Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that is characterized by progressive loss of myocardium that is replaced by fibro-fatty cells, arrhythmias, and sudden cardiac death. While myocardial degeneration and fibro-fatty replacement occur in specific locations, the underlying molecular changes remain poorly characterized. Here, we aim to delineate local changes in gene expression to identify new genes and pathways that are relevant for specific remodelling processes occurring during ACM. METHODS AND RESULTS: Using Tomo-Seq, genome-wide transcriptional profiling with high spatial resolution, we created transmural epicardial-to-endocardial gene expression atlases of explanted ACM hearts to gain molecular insights into disease-driving processes. This enabled us to link gene expression profiles to the different regional remodelling responses and allowed us to identify genes that are potentially relevant for disease progression. In doing so, we identified distinct gene expression profiles marking regions of cardiomyocyte degeneration and fibro-fatty remodelling and revealed Zinc finger and BTB domain-containing protein 11 (ZBTB11) to be specifically enriched at sites of active fibro-fatty replacement of myocardium. Immunohistochemistry indicated ZBTB11 to be induced in cardiomyocytes flanking fibro-fatty areas, which could be confirmed in multiple cardiomyopathy patients. Forced overexpression of ZBTB11 induced autophagy and cell death-related gene programmes in human cardiomyocytes, leading to increased apoptosis. CONCLUSION: Our study shows the power of Tomo-Seq to unveil new molecular mechanisms in human cardiomyopathy and uncovers ZBTB11 as a novel driver of cardiomyocyte loss.

Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Cytoskeleton-Associated Protein 4 as a New Modulator of Fibroblasts Activation.

BACKGROUND: Genome-wide transcriptome analysis has greatly advanced our understanding of the regulatory networks underlying basic cardiac biology and mechanisms driving disease. However, so far, the resolution of studying gene expression patterns in the adult heart has been limited to the level of extracts from whole tissues. The use of tissue homogenates inherently causes the loss of any information on cellular origin or cell type-specific changes in gene expression. Recent developments in RNA amplification strategies provide a unique opportunity to use small amounts of input RNA for genome-wide sequencing of single cells. METHODS: Here, we present a method to obtain high-quality RNA from digested cardiac tissue from adult mice for automated single-cell sequencing of both the healthy and diseased heart. RESULTS: After optimization, we were able to perform single-cell sequencing on adult cardiac tissue under both homeostatic conditions and after ischemic injury. Clustering analysis based on differential gene expression unveiled known and novel markers of all main cardiac cell types. Based on differential gene expression, we could identify multiple subpopulations within a certain cell type. Furthermore, applying single-cell sequencing on both the healthy and injured heart indicated the presence of disease-specific cell subpopulations. As such, we identified cytoskeleton-associated protein 4 as a novel marker for activated fibroblasts that positively correlates with known myofibroblast markers in both mouse and human cardiac tissue. Cytoskeleton-associated protein 4 inhibition in activated fibroblasts treated with transforming growth factor ß triggered a greater increase in the expression of genes related to activated fibroblasts compared with control, suggesting a role of cytoskeleton-associated protein 4 in modulating fibroblast activation in the injured heart. CONCLUSIONS: Single-cell sequencing on both the healthy and diseased adult heart allows us to study transcriptomic differences between cardiac cells, as well as cell type-specific changes in gene expression during cardiac disease. This new approach provides a wealth of novel insights into molecular changes that underlie the cellular processes relevant for cardiac biology and pathophysiology. Applying this technology could lead to the discovery of new therapeutic targets relevant for heart disease.

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.