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

<div><p>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 <i>Bin1</i> 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.</p></div

Cartoon of cardiac bridging integrator 1 (cBIN1)-recruited charged multivesicular body protein 4B (CHMP4B) for microparticle (MP) release from cBIN1-microfolds to extracellular space, responsible for the blood availability of cBIN1.

<p>TT, transverse-tubule.</p

Isolated cardiomyocytes release cardiac bridging integrator 1 (cBIN1) microparticles (MPs).

<p>(A) Western blot data of cBIN1 in cardiomyocyte lysates (positive control), MP pellets, and MP-free supernatant of culture medium. (B) Nanoparticle tracking analysis of resuspended MPs purified from culture medium. (C) Transmission electron microscopy (TEM) images (15,000x) of MPs prepared from cardiomyocyte culture medium after negative staining and immunogold labeling. Left, mouse immunoglobulin G (IgG) control; right, mouse anti-BIN1. Histogram data of cBIN1-MP sizes are also included. (D) Top: spinning-disc confocal images of cardiomyocyte-derived MPs colabeled with annexin V (green) and cBIN1 (red) or IgG control (scale bar, 10 μm). Frequency distribution of cBIN1-MP sizes measured by confocal imaging are shown on the right. Bottom: representative enlarged confocal images of annexin V/cBIN1-MPs (scale bar, 1 μm). Quantification of particle counts with different cBIN1 labeling patterns. (E) Representative stochastic optical reconstruction microscopy (STORM) images of annexin V/cBIN1-MPs (scale bar, 1 μm). Frequency distribution of cBIN1-MP sizes measured by STORM imaging are shown on the right. CM, cardiomyocyte; Conc., concentration; IB, immunoblotting.</p

Cardiac bridging integrator 1 (cBIN1) is present in human plasma and reduced in heart failure.

<p>(A) Western blot (WB) analysis of protein lysates and immunoprecipitation products from normal human heart lysates and plasma. (B) Mass spectrometry analysis of a cBIN1 protein band from human heart lysate immunoprecipitation (IP). (C) Hypotonic shock induced by double-distilled water increases plasma cBIN1 measured by a cBIN1-specific enzyme-linked immunosorbent assay (ELISA), allowing detection of full blood content. Left: final plasma cBIN1 measured by ELISA following increasing amount of water dilution. Red arrow: Maximal concentration was reached at 25% dilution of plasma (1 volume of plasma with 3 volumes of water). Right: final plasma cBIN1 concentration in 25% plasma diluted in physiological buffered saline (PBS), triton detergent buffer (TB), and double-distilled water. (D) Plasma cBIN1 quantified in healthy control patients and patients with heart failure (HF) (<i>n</i> = 10). *** indicates <i>p</i> < 0.001 using a Student <i>t</i> test. Con., control; IgG, immunoglobulin G; SH3, SRC homology 3.</p

Cardiac bridging integrator 1 (cBIN1) in mouse plasma microparticles (MPs) originate from cardiomyocytes.

<p>(A) Transmission electron microscopy (TEM) imaging of cBIN1 in negative-stained and immunogold-labeled MPs (approximately 200 nm) purified from mouse plasma. Scale bar, 200 nm. (B) Frequency distribution of cBIN1-MP sizes measured by TEM imaging. (C) Nanoparticle tracking analysis of resuspended MPs purified from mouse plasma. (D) Flow cytometry quantification of total MP and cBIN1/annexin V-MP concentrations in plasma from mice with cardiac-specific heterozygous <i>Bin1</i> deletion (<i>Bin1</i> HT) and their wild-type (WT) littermate controls. (E) Immunoprecipitation and western blot data of cBIN1 protein in both myocardial tissue and plasma from WT and <i>Bin1</i> HT mice. * and ** indicate <i>p</i> < 0.05 and <i>p</i> < 0.01, respectively, using a Student <i>t</i> test. IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation.</p

Cardiac bridging integrator 1 (cBIN1) recruits charged multivesicular body protein 4B (CHMP4B) through the N-terminal BAR (N-BAR) domain.

<p>(A) Schematics of cBIN1 constructs, including cBIN1 full length (FL) and truncation mutants, the N-BAR domain (BAR), and the C-terminal (CT) domain. (B) Coimmunoprecipitation and western blot results of exogenous cBIN1-V5 and CHMP4B-Flag coexpressed in HeLa cells. (C) Coimmunoprecipitation of exogenous V5-tagged cBIN1 full-length and truncated proteins with CHMP4B-Flag when coexpressed in HeLa cells and immunoprecipitated with anti-Flag antibody. (D) Western blot results from cell lysates and medium MP fraction for HeLa cells overexpressing V5-tagged cBIN1 full length (FL), N-BAR domain (BAR), and CT domain. Cells are from 4 independent experimental repeats. *** indicates <i>p</i> < 0.001 using a one-way ANOVA test. IgG, immunoglobulin G; MBD, Myc-binding domain; SH3, SRC homology 3.</p

Small interfering RNA (siRNA) mediated charged multivesicular body protein 4B (CHMP4B) knockdown reduces cardiac bridging integrator 1 (cBIN1) microparticle (MP) formation and release.

<p>(A) HeLa cells overexpressing cBIN1–green fluorescent protein (GFP) release cBIN1-MPs. Live-cell imaging of HeLa cells expressing cBIN1-GFP with surface labeling of annexin V-Alexa 647. Left, a maximum projection image of a time series of images (every 5 seconds for 2 minutes) of a HeLa cell expressing cBIN1-GFP and surface labeled with annexin V. Right, time-lapse frame images of the boxed area in the left image at the indicated time points. Western blot also identified cBIN1-GFP in MPs from HeLa cell culture medium. (B–D) The effect of CHMP4B siRNA on cBIN1-MP formation and release. (B) Western blot of CHMP4B in HeLa cells treated with CHMP4B siRNA or nontargeting control siRNA. (C) Representative images and quantification of surface annexin V particles in control or CHMP4B siRNA pretreated HeLa cells overexpressing cBIN1-GFP. (D) Western blot results of cBIN1-GFP in cell lysates and medium MP fraction from HeLa cells treated with control or CHMP4B siRNA. Cells are from 3 independent experimental repeats. ** and *** indicate <i>p</i> < 0.05 and <i>p</i> < 0.001, respectively, using an unpaired Student <i>t</i> test. ESCRT, endosomal sorting complexes required for transport; GFP, green fluorescent protein.</p