Introduction: Cryoinjury has been frequently used as an alternative approach to mimic myocardial infarction in various in vivo studies. This model, often performed in rodents or zebrafish, was shown to be representative of myocardial infarctions encountered in clinical practice. Here, we performed cryoinjury on rat living myocardial slices. These are ultrathin sections of cardiac tissue, that maintain the native multicellularity, architecture, and structure of the heart while preserving tissue viability in the absence of coronary perfusion in vitro due to free diffusion of oxygen and nutrients into their innermost cells, preventing ischaemic damage and allowing for chronic culture.

Purpose: The aim of this study is to establish a 3D model of myocardial infarction in living myocardial slices using cryoinjury usable for ex vivo drug testing.

Methods: Living myocardial slices were generated from rat left ventricular tissue and were subjected to cryoinjury to damage 30% of their length. Cryoinjured slices, along with control slices, were cultured while electrically stimulated and mechanically stretched. After 24 hours in culture, force measurements of myocardial slices were performed (0.2Hz, 30V, 7ms). Histological studies were done to assess tissue viability, fibrosis markers and cardiomyocyte cross-sectional area. Eventually, slices were cut into two parts: peri-injury (including the cryoinjury with 1mm margins of neighboring tissue) and remote region. Both regions, along with supernatant, were then analyzed for cardiac remodeling marker genes and transcripts applying real-time PCR, western blot and mass spectrometry-based protein profiling.

Results: Cell viability assessment using live/dead cell staining demonstrated complete death of cells in the cryoinjured area of myocardial slices. Measurement of contractile force of cryoinjured slices after 24 hours in culture revealed reduced maximal contractility and slower kinetics, including time to peak, time to relaxation and decay rate compared to control slices, indicating that contractile function of myocardial slices deteriorated significantly following cryoinjury. Quantification of cardiomyocyte cross-sectional area demonstrated hypertrophy of cardiomyocytes in the remote region of cryoinjured slices compared to both the peri-injury area and the control slices. Gene expression analysis of myocardial slices highlighted an increase in fibroblast gene activation in the remote region, as well as an increased expression of inflammation markers in the peri-injury region of myocardial slices compared to control. Proteomics analysis of myocardial slices demonstrated upregulation of proteins associated with metabolic pathways, dilated cardiomyopathy, hypertrophic cardiomyopathy, and cardiac muscle contraction in cryoinjured slices compared to control. In line, enhanced abundancy of proteins and microRNAs associated with fibrosis and cardiac healing was found in the supernatant of cryoinjured slices, indicating a change in the secretome.

Conclusions: Based on contractile, structural and multi-OMICs analysis, we here report a reproducible model of myocardial infarction in living myocardial slices. This 3D model could be utilized for investigating various aspects of cardiac biology, such as electrophysiology, biochemistry and molecular biology, in addition to high applicability in novel drug discovery and regenerative medicine.