Extracellular vesicles do not contribute to higher circulating levels of soluble LRP1 in idiopathic dilated cardiomyopathy

Abstract

Idiopathic dilated cardiomyopathy (IDCM) is a frequent cause of heart transplantation. Potentially valuable blood markers are being sought, and low-density lipoprotein receptor-related protein 1 (LRP1) has been linked to the underlying molecular basis of the disease. This study compared circulating levels of soluble LRP1 (sLRP1) in IDCM patients and healthy controls and elucidated whether sLRP1 is exported out of the myocardium through extracellular vesicles (EVs) to gain a better understanding of the pathogenesis of the disease. LRP1 α chain expression was analysed in samples collected from the left ventricles of explanted hearts using immunohistochemistry. sLRP1 concentrations were determined in platelet-free plasma by enzyme-linked immunosorbent assay. Plasma-derived EVs were extracted by size-exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis and cryo-transmission electron microscopy. The distributions of vesicular (CD9, CD81) and myocardial (caveolin-3) proteins and LRP1 α chain were assessed in SEC fractions by flow cytometry. LRP1 α chain was preferably localized to blood vessels in IDCM compared to control myocardium. Circulating sLRP1 was increased in IDCM patients. CD9- and CD81-positive fractions enriched with membrane vesicles with the expected size and morphology were isolated from both groups. The LRP1 α chain was not present in these SEC fractions, which were also positive for caveolin-3. The increase in circulating sLRP1 in IDCM patients may be clinically valuable. Although EVs do not contribute to higher sLRP1 levels in IDCM, a comprehensive analysis of EV content would provide further insights into the search for novel blood markers.

Keywords: biomarker; extracellular vesicles; idiopathic dilated cardiomyopathy; sLRP1; size-exclusion chromatography.

Figure 1

Analysis of myocardial LRP1 α chain expression and localization. Representative confocal microscope images showing specific global detection of LRP1 α chain (red) (A) and more detailed localization of LRP1 α chain (green) in IsoB4—(grey) (B), CD31—(grey) (C and D) and vWF—(grey) (E) positive vessels. Cardiac muscle and cell nuclei are counterstained using an anti‐cTnI antibody (red) and DAPI (blue), respectively. Scale bars = 50 μm (F) Histogram represents quantification of LRP1 α chain positivity as percentage of arbitrary units per area. *P = 0.02.

Figure 2

Assessment of myocardial lipid content. Distribution of cholesteryl esters (CE), triglycerides (TG) and free cholesterol (FC) in both groups (St = standard, C = control and D = dilated cardiomyopathy). Histograms represent the quantification of total TG, CE and FC. n = 5 each control and idiopathic dilated cardiomyopathy (IDCM). *P = 0.026.

Figure 3

Increased levels of circulating sLRP1 in idiopathic dilated cardiomyopathy (IDCM) patients. The histogram represents quantification of circulating sLRP1 by ELISA. *P = 0.034. n = 15 control and n = 20 IDCM.

Figure 4

sLRP1 is not found in platelet‐free plasma‐derived extracellular vesicles (EV) fractions. (A) Representative analyses of platelet‐free plasma‐derived size‐exclusion chromatography (SEC) fractions by flow cytometry. Median fluorescence intensity (MFI) was measured, and the isotype negative control is represented as a dashed line. (B) Electron micrographs of vesicular protein‐enriched (8–10) and non‐enriched (11–13) fractions from a healthy control (left) and a idiopathic dilated cardiomyopathy (IDCM) patient (right) showing multifaceted assembly of round electron‐lucent membrane vesicles and a large quantity of smaller electron‐dense structures lacking a membrane bilayer, respectively. (C) Characteristic particle size distribution and concentration by NTA (healthy control, left; IDCM, right). n = 5 each control and IDCM. Scale bars = 100 nm.