Essential role of Alix in regulating cardiomyocyte exosome biogenesis under physiological and stress conditions
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Updated: Jun 28, 2025
Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice
Published on: April 10, 2019
Xinjian Wang1, Shuxian Han1, Jinxiu Liang1
1Center for Genetic Medicine, the Fourth Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China; Institute of Genetics, Zhejiang University International School of Medicine, Hangzhou, Zhejiang, China; Division of Medical Genetics and Genomics, The Children's Hospital, Zhejiang University School of Medicine and National Clinical Research Center for Child Health, Hangzhou, Zhejiang, China; Zhejiang Provincial Key Laboratory of Genetic & Developmental Disorders, Hangzhou, Zhejiang, China.
Insights
Alix is crucial for cardiac exosome biogenesis, regulating intercellular communication in the heart. Its depletion impairs exosome production, while stress increases Alix and exosome levels, highlighting its role in cardiovascular health.
Area of Science:
- Cardiovascular Biology
- Cell Biology
- Molecular Medicine
Background:
- Exosomes from cardiomyocytes mediate heart intercellular communication.
- Exosomal components are linked to cardiovascular diseases.
- The role of the ESCRT machinery and Alix in cardiomyocyte exosome biogenesis is unclear.
Purpose of the Study:
- To investigate the role of Alix in cardiomyocyte exosome biogenesis.
- To determine the impact of Alix depletion on cardiac exosome production.
- To explore Alix-mediated exosome biogenesis under cardiac stress.
Main Methods:
- Super-resolution imaging of Alix and MVBs in cardiomyocytes.
- Generation of cardiomyocyte-specific Alix-knockout mice and Alix-knockdown H9c2 cells.
- Exosome isolation and characterization (Western blot, NTA, TEM).
- Analysis of ESCRT machinery and MVB formation.
- Cardiac pressure overload model (TAC) to study stress response.
Main Results:
- Alix localizes to the MVB membrane in cardiomyocytes.
- Alix deletion or knockdown reduces plasma and cellular exosome content.
- Alix depletion impairs ESCRT machinery and MVB formation, compromising exosome biogenesis.
- Cardiac pressure overload increases Alix, MVBs, and exosome levels, which is prevented by Alix deletion.
Conclusions:
- Alix is an essential regulator of cardiac exosome biogenesis.
- Alix function in exosome production is sensitive to cardiac stress.
- Findings suggest Alix as a potential therapeutic target for cardiovascular diseases.
Background:
Exosomes released by cardiomyocytes are essential mediators of intercellular communications within the heart, and various exosomal proteins and miRNAs are associated with cardiovascular diseases. However, whether the endosomal sorting complex required for transport (ESCRT) and its key component Alix is required for exosome biogenesis within cardiomyocyte remains poorly understood.
Methods:
Super-resolution imaging was performed to investigate the subcellular location of Alix and multivesicular body (MVB) in primary cardiomyocytes. Cardiomyocyte-specific Alix-knockout mice were generated using AAV9/CRISPR/Cas9-mediated in vivo gene editing. A stable Alix-knockdown H9c2 cardiomyocyte line was constructed through lentiviral-mediated delivery of short hairpin RNA. In order to determine the role of Alix in controlling exosome biogenesis, exosomes from cardiomyocyte-specific Alix-knockout mice plasma and Alix-knockdown H9c2 culture medium were isolated and examined by western blot, NTA analysis and transmission electron microscopy. Biochemical and immunofluorescence analysis were performed to determine the role of ESCRT machinery in regulating MVB formation. Lastly, transverse aortic constriction (TAC)-induced cardiac pressure overload model was established to further explore the role of Alix-mediated exosome biogenesis under stress conditions.
Results:
A significant proportion of Alix localized to the MVB membrane within cardiomyocytes. Genetic deletion of Alix in murine heart resulted in a reduction of plasma exosome content without affecting cardiac structure or contractile function. Consistently, the downregulation of Alix in H9c2 cardiomyocyte line also suppressed the biogenesis of exosomes. We found the defective ESCRT machinery and suppressed MVB formation upon Alix depletion caused compromised exosome biogenesis. Remarkably, TAC-induced cardiac pressure overload led to increased Alix, MVB levels, and elevated plasma exosome content, which could be totally abolished by Alix deletion.
Conclusion:
These results establish Alix as an essential and stress-sensitive regulator of cardiac exosome biogenesis and the findings may yield valuable therapeutic implications.
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