Proteome characterisation of extracellular vesicles isolated from heart
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Updated: Nov 9, 2025
Author Spotlight: Advancing the Analysis of Plasma Extracellular Vesicle Proteome for Cardiovascular Biomarker Studies
Published on: January 31, 2025
Bethany Claridge1,2, Alin Rai2,3,4, Haoyun Fang2
1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, Victoria, Australia.
Insights
Researchers developed a new method to isolate cardiac extracellular vesicles (cEVs) from mouse hearts. This technique provides insights into cEV protein composition and potential roles in cardiovascular health and disease.
Area of Science:
- Cardiovascular Biology
- Extracellular Vesicle Research
- Proteomics
Background:
- Cardiac intercellular communication is vital for heart function but dysregulated in cardiovascular diseases.
- Cardiac extracellular vesicles (cEVs) are key signaling mediators, yet their isolation and characterization are challenging.
- Understanding cEV protein composition is crucial for elucidating their role in cardiac physiology and pathology.
Purpose of the Study:
- To develop and validate a robust method for isolating cardiac extracellular vesicles (cEVs) from mouse hearts.
- To characterize the protein content of isolated cEVs using mass spectrometry.
- To investigate the potential functions and cellular origins of cEVs.
Main Methods:
- Enzymatic perfusion (Langendorff-collagenase) and differential centrifugation for cEV isolation from mouse hearts.
- Characterization of cEVs using nanoparticle tracking analysis and Western blotting for EV markers.
- Proteomic profiling of cEVs via mass spectrometry.
Main Results:
- Isolated cEVs (∼200 nm) expressed classical EV markers and were depleted of blood and cardiac damage proteins.
- Proteomic analysis identified 1721 proteins in cEVs, involved in proteostasis, metabolism, and EV biogenesis.
- cEVs contained markers from diverse cardiac and immune cells, suggesting multifaceted origins and functions.
Conclusions:
- A reliable method for cEV isolation from the heart was established.
- The proteomic profile of cEVs suggests roles in metabolic regulation and intercellular signaling.
- These findings pave the way for further research into cEVs in cardiac disease.
Abstract:
Cardiac intercellular communication is critical for heart function and often dysregulated in cardiovascular diseases. While cardiac extracellular vesicles (cEVs) are emerging mediators of signalling, their isolation remains a technical challenge hindering our understanding of cEV protein composition. Here, we utilised Langendorff-collagenase-based enzymatic perfusion and differential centrifugation to isolate cEVs from mouse heart (yield 3-6 μg/heart). cEVs are ∼200 nm, express classical EV markers (Cd63/81/9+ , Tsg101+ , Pdcd6ip/Alix+ ), and are depleted of blood (Alb/Fga/Hba) and cardiac damage markers (Mb, Tnnt2, Ldhb). Comparison with mechanically-derived EVs revealed greater detection of EV markers and decreased cardiac damage contaminants. Mass spectrometry-based proteomic profiling revealed 1721 proteins in cEVs, implicated in proteasomal and autophagic proteostasis, glycolysis, and fatty acid metabolism; essential functions often disrupted in cardiac pathologies. There was striking enrichment of 942 proteins in cEVs compared to mouse heart tissue - implicated in EV biogenesis, antioxidant activity, and lipid transport, suggesting active cargo selection and specialised function. Interestingly, cEVs contain marker proteins for cardiomyocytes, cardiac progenitors, B-cells, T-cells, macrophages, smooth muscle cells, endothelial cells, and cardiac fibroblasts, suggesting diverse cellular origin. We present a method of cEV isolation and provide insight into potential functions, enabling future studies into EV roles in cardiac physiology and disease.
