Impact of early pericardial fluid chymase activation after cardiac surgery

Abstract

Introduction: Chymase is a highly destructive serine protease rapidly neutralized in the circulation by protease inhibitors. Here we test whether pericardial fluid (PCF) chymase activation and other inflammatory biomarkers determine intensive care unit length of stay, and explore mechanisms of chymase delivery by extracellular vesicles to the heart.

Methods: PCF was collected from adult patients (17 on-pump; 13 off-pump) 4 h after cardiac surgery. Extracellular vesicles (EVs) containing chymase were injected into Sprague-Dawley rats to test for their ability to deliver chymase to the heart.

Results: The mean intensive care unit (ICU) stay and mean total length of stay was 2.17 ± 3.8 days and 6.41 ± 1.3 days respectively. Chymase activity and 32 inflammatory markers did not differ in on-pump vs. off-pump cardiac surgery. Society of Thoracic Surgeons Predicted Risk of Morbidity and Mortality Score (STS-PROM), 4-hour post-surgery PCF chymase activity and C-X-C motif chemokine ligand 6 (CXCL6) were all independent predictors of ICU and total hospital length of stay by univariate analysis. Mass spectrometry of baseline PCF shows the presence of serine protease inhibitors that neutralize chymase activity. The compartmentalization of chymase within and on the surface of PCF EVs was visualized by immunogold labeling and transmission electron microscopy. A chymase inhibitor prevented EV chymase activity (0.28 fmol/mg/min vs. 14.14 fmol/mg/min). Intravenous injection of PCF EVs obtained 24 h after surgery into Sprague Dawley rats shows diffuse human chymase uptake in the heart with extensive cardiomyocyte damage 4 h after injection.

Discussion: Early postoperative PCF chymase activation underscores its potential role in cardiac damage soon after on- or off-pump cardiac surgery. In addition, chymase in extracellular vesicles provides a protected delivery mechanism from neutralization by circulating serine protease inhibitors.

Keywords: STS-PROM; cardiovascular surgery; chymase; exosomes; extracellular vesicles; inflammation; length of stay; pericardial fluid.

Figure 1

Chymase activity (A) and angiotensin II peptide concentration (B) measured in pericardial fluid (n = 5) upon opening of the pericardium (time 0), 4, 12, and 24 h time points after cardiopulmonary bypass. Chymase activity is calculated based on chymase-mediated Ang-(1–12) metabolism. Representative western blot (C) of human chymase in an equal volume of PCF obtained from the same patient (1 of the 5) at documented time points in the other panels (time 0, 4, 12, and 24 h after cardiopulmonary bypass). 05; **p < 0.01; ***p < 0.001 vs. time 0. Box and whisker plots (median, interquartile range with min and max values). Mean indicated by red diamond).

Figure 2

Immunogold ultrathin transmission electron microscopy of microvesicles at time 0. Immunogold TEM using gold beads bound to chymase, CD9 or Annexin V antibodies. Chymase is found inside vesicles (A, D2, E) but is predominantly on the membrane surface (B, D1, D2, E) as indicated by co-labeling for the tetraspanin CD9 (yellow arrowheads) or Annexin V (red arrowhead). The omission of the chymase antibody shows no staining in the negative sections (C,F). Scale bars 0.5 μm - 25,000X (B, C), 0.2 μm - 50,000X (inset D2) and 0.1 μm -100,000X (A, D, inset D1, E,F).

Figure 3

Naïve saline injected rat (A) and a rat 4 h after injection with PCF microvesicles obtained from a patient (24 h after cardiac surgery) demonstrate human chymase (red) (B, C) in the interstitium and within endothelial cells (B-white box inset of C) in the rat heart. DAPI nuclei: Blue, von Willebrand Factor: Green (endothelium). The red outline around the vessel is the autofluorescence of the internal elastic membrane. Red blood cells auto fluoresce with a subdued yellowish tinge (within a vessel in A). Scale bar = 20 µm.

Figure 4

H&E staining of naïve saline injected rat heart (A) and four and 24 h after injection of extracellular vesicles (B,C). The normal heart (A) demonstrates homogeneous H&E staining and compactly spaced cardiomyocytes. Microvesicle-injected hearts demonstrate increased interstitial space and diffuse breakdown of cardiomyocytes and myosin (B, 20X). At 40x, there is myocyte loss, myosin breakdown, and damaged capillary and endothelial cells (arrow) consistent with the increased endothelial chymase in (C).