Endomyocardial biopsy-derived fibroblasts as a screening tool to distinct heart failure with reduced and preserved ejection fraction

Introduction

Accumulating evidence states the involvement of cardiac fibroblasts (CFs) in heart failure, a leading cause of death with a mortality rate of approximately 50 % within five years of diagnosis. Despite extensive studies, anti-inflammatory and anti-fibrotic treatment strategies have not been successful for heart failure so far. This indicates the need to better assess/diagnose the ongoing inflammatory and fibrosis process in the heart and the need of patient-specific model systems to screen for potential anti-inflammatory/anti-fibrotic treatment options. Due to the specific role of fibroblasts in pathological cardiac tissue remodeling by defining extracellular matrix composition and boosting inflammatory reactions, we propose that CFs can be used as a diagnostic tool to assess patient- and patient-group specific cardiac inflammation and fibrosis/remodeling. We here compared CFs from heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF) patients.

Aim

This work aimed to elucidate whether human endomyocardial biopsy-derived CFs can be used to discriminate patients on single patient level as well as on patient-group (HFpEF versus HFrEF patients) level based on presented methods. 

Methods

Basal characterization of HFpEF- versus HFrEF-derived CFs passaged in FBS-supplemented (5 % FBS + 5 % HS) and xeno-free cell culture media was performed analyzing cell amount, viability, and collagen deposition (Crystal Violet Assay, MTT and Sirius Red Assay, respectively) and gene expression analysis (qRT-PCR and scRNA-Sequencing). 

Testing hCFs responsiveness to TGF-β1 stimulation, the suitability of the cultured CFs as a screening platform for patient characterization was assessed by high-content screening of immunofluorescence-stained human CFs.

Results

Passaged HFpEF-derived CFs showed higher cell amount, viability, collagen deposition and overall metabolism as compared to HFrEF-derived CFs under basal conditions. HFpEF- and HFrEF-derived CFs present separated cell clusters based single-cell RNA sequencing. HFpEF-derived CFs showed higher expression of cardiac inflammation-associated-genes such as chemokines and cytokines (CXCL1, CXCL3, IL-6) or NLRP3-activity corresponding gene expression as compared to HFrEF-derived CFs, which were characterized by higher integrin expression. High-Content screening of patient-derived CFs showed different regulation of fibroblast-characterizing markers such as α-SMA, FSP-1 and FAP, as well as collagen-3 and NLRP3 inflammasome activity markers between both patient-groups under basal and TGF-β1 stimulation conditions. Abovementioned findings were observed in hCFs cultured in FBS-supplemented medium and confirmed under xeno-free conditions. 

Conclusion

Here, we demonstrate that HFpEF- and HFrEF-derived CFs can be distinguished based on functional characteristics in vitro as well as on their gene expression. Additionally, we are presenting a novel platform to screen immunofluorescence-stained human CFs to assess patient- as well as patient-group specific marker expression levels in a high-content manner.