Essential role of Alix in regulating cardiomyocyte exosome biogenesis under physiological and stress conditions

Xinjian Wang; Shuxian Han; Jinxiu Liang; Chen Xu; Ranran Cao; Shuoyang Liu; Yi Luan; Ying Gu; Peidong Han

doi:10.1016/j.yjmcc.2024.04.001

Essential role of Alix in regulating cardiomyocyte exosome biogenesis under physiological and stress conditions

J Mol Cell Cardiol. 2024 Apr 8:190:35-47. doi: 10.1016/j.yjmcc.2024.04.001. Online ahead of print.

Authors

Xinjian Wang¹, Shuxian Han¹, Jinxiu Liang¹, Chen Xu¹, Ranran Cao¹, Shuoyang Liu¹, Yi Luan², Ying Gu¹, Peidong Han³

Affiliations

¹ Center 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.
² Department of Cardiology, Key Laboratory of Cardiovascular Intervention and Regenerative Medicine of Zhejiang Province, Sir Run Run Shaw Hospital, Zhejiang University, School of Medicine, Hangzhou, Zhejiang 310016, China.
³ Center 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. Electronic address: hanpd@zju.edu.cn.

PMID: 38593639
DOI: 10.1016/j.yjmcc.2024.04.001

Abstract

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.

Keywords: Cardiomyocyte; ESCRT; Exosome; Multivesicular body; Somatic mutagenesis.