Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Ferroptosis: a key player in the ex-vivo cryoinjury model in living myocardial slices
N. Abbas1, F. Waleczek1, F. Perbellini1, A. Pich2, M. Fuchs3, A. Pfanne1, A. Just1, J. Fiedler4, T. Thum1, for the study group: IMTTS
1Institut für Molekulare und Translationale Therapiestrategien, OE-8886, Medizinische Hochschule Hannover, Hannover; 2Institute of Toxicology and Core Unit Proteomics, Hannover; 3Fraunhofer Institute of Toxicology and Experimental Medicine (ITEM), Hannover; 4Kardiovaskuläre Forschung, C11, Fraunhofer-Institut für Toxikologie und Experimentelle Medizin ITEM, Hannover;

Introduction: Cryoinjury (CI) is frequently used as an alternative approach to mimic myocardial infarction (MI) in in vivo models and was shown to be representative of MI encountered in clinical practice. Here, rat living myocardial slices (LMS), which are ultrathin sections of cardiac tissue with maintained multicellularity and heart architecture, were subjected to CI to investigate the mechanisms underlying cardiac injury.

Ferroptosis, an iron-dependent form of cell death, was found to be a potential key player in LMS after injury. This form of cell death does not exhibit typical characteristics of programmed cell death, and is mediated by impaired iron metabolism and lipid peroxidation signaling. Here, we investigate ferroptosis inhibition as a possible novel therapeutic approach against cardiovascular disease.

Purpose: The aim of this study is to establish an ex-vivo model of myocardial injury in LMS and to identify the role of ferroptosis as an underlying mechanism and potential therapeutic target.

Methods: LMS were generated from left ventricle heart tissue samples from rats and were subjected to CI damage comprising 30% of their total area. CI- and control-LMS were cultivated with and without inhibition of ferroptosis using Fer-1. After 24 hours, the contractile function was assessed and histological studies were done to quantify tissue fibrosis and cardiomyocyte cross-sectional area (CM-CSA). Additionally, the expression of cardiac remodeling marker genes was measured by applying RT-qPCR, western blot (WB) and mass spectrometry-based protein profiling. An NF-κB reporter assay monitoring inflammatory responses was performed using HEK-293T cells, and Fer-1 treatment of H9C2 cells was performed, followed by protein detection.

Results: Force measurements revealed reduced maximal contractility and slower kinetics after CI, indicating functional impairment. Quantification of CM-CSA demonstrated cellular hypertrophy in regions remote to CI. Furthermore, gene expression and proteomics analysis highlighted the enrichment of pathways associated with fibroblast activation, inflammation and cardiomyopathies in CI-LMS, whereas secretome analysis demonstrated an enhanced abundancy of proteins and microRNAs associated with fibrosis and cardiac healing. Taken together, these data indicate that our model successfully mimics characteristic features of cardiac injury in vivo.

Multi-dimensional in silico analysis revealed an enhanced abundancy of factors associated with ferroptosis in CI-LMS, e.g. transferrin, glutathione peroxidase 3, and heme oxygenase (HMOX), thereby hinting for possible causality. Pharmacological blocking of ferroptosis was able to alleviate the functional impairment in CI-LMS and promote the expression of HMOX1 – a known antioxidant enzyme - in H9C2 cells. In line, NF-κB reporter assay underlined a dose dependent effect of Fer-1 treatment on NF-κB signaling, indicating an additional anti-inflammatory effect further supporting cardiac healing.

Conclusions: Based on contractile, structural, and multi-OMICs analysis, we report a novel ex-vivo model of cardiac injury in LMS. We found that the recently identified iron-dependent cell death mechanism, ferroptosis, was activated in our model. Therapeutic inhibition improved cardiac function, halted oxidative stress and triggered anti-inflammatory effects. These findings suggest that targeting endogenous ferroptosis is a possible strategy for treating deadly ischemic heart disease.


https://dgk.org/kongress_programme/jt2022/aP1976.html