Abstract 604 Single-nuclei RNA sequencing identifies putative targets to counteract fibroblast activation in patients with chronic cardiac disease

Background: Although cardiac fibrosis is a major driver of heart failure progression associated with poor prognosis, no anti-fibrotic treatments are available in patients yet. In this study, we analyzed cardiac fibroblasts of patients with chronic heart disease on a single-cell resolution to gain insights into their contribution to maladaptive fibrotic remodeling and to identify novel therapeutic targets.

Methods: Transcriptome profiling of patients’ cardiac fibroblasts was conducted using single-nuclei RNA sequencing of healthy (2 samples), hypertrophic (5 samples), and failing (2 samples) human heart tissue. Unsupervised clustering defined distinct fibroblast clusters. Molecular signatures of healthy and diseased fibroblasts were obtained by gene ontology analysis of differentially expressed genes.

Results: Both, hypertrophic and failing heart tissue demonstrated an increased proportion of pathological fibroblast subclusters concomitant with a transcriptional shift towards profibrotic gene signatures during chronic cardiac disease. Importantly, we identified a particular fibroblast population featuring molecular characteristics of activated fibroblasts in these patients. Besides a high expression of POSTN and COL1A1, this subcluster showed a specific upregulation of adipocyte enhancer-binding protein 1 (AEBP1), which is known for inducing adipocyte proliferation and is involved in the regulation of collagen fibrillogenesis. The induction of AEBP1 in activated fibroblasts was confirmed in vitro upon TGFbeta stimulation (1.6 ± 0.2-fold induction, p < 0.05). As AEBP1 expression is upregulated in activated fibroblasts of patients with hypertrophic and failing hearts, we further examined the effect of siRNA-mediated AEBP1 silencing as a possible therapeutic approach on the hallmark capabilities of human cardiac fibroblasts. Indeed, AEBP1 silencing resulted in a significant reduction of fibroblasts proliferation, migration, and contractile capacity (all p < 0.05). Furthermore, we observed attenuated stress marker gene expression such as ACTA2 and COL3A1 and alphaSMA-formation after AEBP1 silencing. Conversely, addition of recombinant AEBP1 to human cardiac fibroblasts augmented contraction (1.3 ± 0.06-fold induction, p < 0.05) and migration (1.2 ± 0.03-fold induction, p < 0.05) in vitro.

Conclusion: Taken together, single-cell profiling of cardiac fibroblasts reveals pathological fibroblast activation in patients with hypertrophic and failing hearts, which might be modulated by AEBP1 silencing as a novel therapeutic approach.

Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5
Single-nuclei RNA sequencing identifies putative targets to counteract fibroblast activation in patients with chronic cardiac disease
F. Böckling¹, M. Shumliakivska², L. Tombor², T. Rasper², L. Nicin², W. Abplanalp², K. Schmitz², D. C. Carstens², T. Holubec³, M. Arsalan³, F. Emrich³, T. Walther³, D. John², A. M. Zeiher², S. Dimmeler², B. Kattih¹
¹Med. Klinik III - Kardiologie Zentrum der Inneren Medizin, Universitätsklinikum Frankfurt, Frankfurt am Main; ²Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration, Goethe Universität Frankfurt am Main, Frankfurt am Main; ³Klinik für Thorax-, Herz- und Thorakale Gefäßchirurgie, Universitätsklinikum Frankfurt, Frankfurt am Main;
Background: Although cardiac fibrosis is a major driver of heart failure progression associated with poor prognosis, no anti-fibrotic treatments are available in patients yet. In this study, we analyzed cardiac fibroblasts of patients with chronic heart disease on a single-cell resolution to gain insights into their contribution to maladaptive fibrotic remodeling and to identify novel therapeutic targets. Methods: Transcriptome profiling of patients’ cardiac fibroblasts was conducted using single-nuclei RNA sequencing of healthy (2 samples), hypertrophic (5 samples), and failing (2 samples) human heart tissue. Unsupervised clustering defined distinct fibroblast clusters. Molecular signatures of healthy and diseased fibroblasts were obtained by gene ontology analysis of differentially expressed genes. Results: Both, hypertrophic and failing heart tissue demonstrated an increased proportion of pathological fibroblast subclusters concomitant with a transcriptional shift towards profibrotic gene signatures during chronic cardiac disease. Importantly, we identified a particular fibroblast population featuring molecular characteristics of activated fibroblasts in these patients. Besides a high expression of POSTN and COL1A1, this subcluster showed a specific upregulation of adipocyte enhancer-binding protein 1 (AEBP1), which is known for inducing adipocyte proliferation and is involved in the regulation of collagen fibrillogenesis. The induction of AEBP1 in activated fibroblasts was confirmed in vitro upon TGFbeta stimulation (1.6 ± 0.2-fold induction, p < 0.05). As AEBP1 expression is upregulated in activated fibroblasts of patients with hypertrophic and failing hearts, we further examined the effect of siRNA-mediated AEBP1 silencing as a possible therapeutic approach on the hallmark capabilities of human cardiac fibroblasts. Indeed, AEBP1 silencing resulted in a significant reduction of fibroblasts proliferation, migration, and contractile capacity (all p < 0.05). Furthermore, we observed attenuated stress marker gene expression such as ACTA2 and COL3A1 and alphaSMA-formation after AEBP1 silencing. Conversely, addition of recombinant AEBP1 to human cardiac fibroblasts augmented contraction (1.3 ± 0.06-fold induction, p < 0.05) and migration (1.2 ± 0.03-fold induction, p < 0.05) in vitro. Conclusion: Taken together, single-cell profiling of cardiac fibroblasts reveals pathological fibroblast activation in patients with hypertrophic and failing hearts, which might be modulated by AEBP1 silencing as a novel therapeutic approach.
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