The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles.

Cecilia Lo,Bing Xu,Yan Liu,Rachel Baum,Robert C Wirka,Robin M Shaw,Tingting Hong,Ying Fu,Kang Zhou,Sosse Agvanian

doi:10.1371/journal.pbio.2002354

Abstract

Microparticles (MPs) are cell–cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.

Highlights

Microparticles (MPs) are cell-derived membrane vesicles that are formed by outward blebbing of the plasma membrane followed by membrane fission and subsequent release of lipid vesicles into circulation
We show that heart ventricular muscle cells, which form the main pumping chambers of the heart, release microparticles in both mouse and human
Ventricular microparticles arise from surface membrane microdomains organized by cardiac bridging integrator 1, a membrane deformation protein that has been shown to be reduced during human heart failure

Summary

Introduction

Microparticles (MPs) are cell-derived membrane vesicles that are formed by outward blebbing of the plasma membrane followed by membrane fission and subsequent release of lipid vesicles into circulation (see reviews in [1,2]). MPs are smaller than apoptotic bodies, which are 1–5 μm, yet larger than exosomes, which are smaller than 100 nm. MP vesicles range between 100 to 1000 nm and, unlike apoptotic bodies, have an impermeable membrane. While exosomes are cytoplasmic in origin and released through exocytosis, MPs are directly derived from the plasma membrane. A wide variety of cell types have been reported to generate MPs, including endothelial cells, vascular smooth muscle cells, and blood cells such as erythrocytes, leukocytes, and platelets [3]. MPs are poised to influence the systemic response in various diseases, such as cancer, inflammatory, autoimmune, and cardiovascular diseases [2,4,5,6]

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