Investigating the mechanisms of small extracellular vesicles in cardiovascular disease using the living myocardial slice platform

Figures

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  Figure 1. Biogenesis pathway of sEVs. sEVs are formed by the initial inward budding of the plasma membrane, generating the early endosomes. Bioactive molecules in the intracellular space are enclosed into late endosomes, forming intraluminal vesicles (ILVs) and packaged in multivesicular bodies (MVBs). The sEVs are released in the extracellular space by exocytosis to reach the recipient cell. Uptake mechanisms include phagocytosis, fusion, or direct binding with plasma membrane receptors. Adapted with permission from “Biogenesis and Cellular uptake of EVs” by Yuanwang Jia, Li Yu, Tieliang Ma, Wenrong Xu, Hui Qian, Yaoxiang Sun and Hui Shi, licensed under CC BY 4.0.

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  Figure 2. sEV-mediated regulation of biological processes. sEVs are produced by all cardiac cell types. Their content mediates intercellular communication, which results in the influence of several pathways, including the proliferation, inflammation, fibrosis, apoptosis, and angiogenesis. Arrows indicate the direction of sEVs released from cardiomyocytes (red), endothelial cells (blue), and fibroblasts (orange).

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  Figure 3. Generation process of the human LMS model. The human donor heart is dissected to retrieve an LV block of 1 cm3. The vibratome precision-cuts the block to obtain layers of 300 μm thickness. The slice is aligned under the microscope for the positioning of the PLA rings perpendicular to the muscle fibers. The LMS is ready to culture.

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  Figure 4. Overview of sEV isolation and characterisation methods. The LMS conditioned media is collected from the culture chamber. The sEV isolation is achieved via ultrafiltration and SEC. The isolated sEVs are characterised based on quantity and s size distribution, marker expression, and shape. Representative images of the output of NTA, NanoFCM, ExoView, and images of sEVs visualized under Transmission Electron Microscopy. “ExoView” reproduced with permission from “Representative ExoView images of indicated immunophenotypically captured and detected Serum 10 k xg EVs” and “Transmission Electron Microscopy” adapted with permission from “Quality control of EVs utilised for comparative NTA device analysis” by Daniel Bachurski, Maximiliane Schuldner, Phuong-Hien Nguyen, Alexandra Malz, Katrin S Reiners, Patricia C Grenzi, Felix Babatz, Astrid C Schauss, Hinrich P Hansen, Michael Hallek and Elke Pogge von Strandmann, licensed under CC BY-NC 4.0.

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  Figure 5. Setup of the air-liquid interface of sEVs treatment on LMS. Media is removed from the culture chamber to expose the top surface of the LMS to air. The sEVs are applied on the surface for their absorption by the LMS. Electrical stimulation, media circulation, and oxygenation are interrupted. Following absorption, media is added to achieve the desired volume and electrical stimulation, media circulation, and oxygenation are resumed.

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  Figure 6. Overview of functional, structural, and biochemical studies on LMS treated with sEVs. The investigation of differential miRNA expression on sEVs can be performed by miRNA arrays. Following the treatment of LMS with cardiac sEVs, functional studies (contractility, live Ca2+ imaging, and conduction velocity), structural studies, and biochemical studies can be carried out on the LMS. Contractility recording adapted with permission from “Contractility recordings and representative confocal images of living myocardial slice” by Filippo Perbellini and Thomas Thum, licensed under CC BY-NC. Live Ca2+ adapted with permission from “Viability in freshly sliced LMS and after 24h of culture” by Eef Dries, Ifigeneia Bardi, Raquel Nunez-Toldra, Bram Meijlink and Cesare M. Terracciano, licensed under CC BY 4.0. Conduction velocity reproduced with permission from “Conduction, connexins, β-adrenergic stimulation and arrhythmogenesis in rat myocardial slices cultured for 24 h. a, b Representative images of longitudinal and transverse conduction maps of a 0 h rat myocardial slice. Black arrows show myocardial fibre direction. Yellow star = location of the stimulating electrode. Area of conduction…