Combined immunoelectron microscopic and computer-assisted image analyses to detect advanced glycation end-products in human myocardium

Abstract

Advanced glycation end-products (AGEs) result from oxidation-reduction reactions that ensue when a sugar becomes adducted to a protein. AGEs cause various complications of diabetes mellitus (DM). Experimental and clinical evidence suggest that AGEs also contribute to the complications of hypertension (HTN). Little is known about the abundance and localization of AGEs in human myocardium. In a few light microscopic studies, the AGE carboxymethyl lysine (CML) has been immunolabeled and localized virtually exclusively to the walls of small arteries. To more precisely delineate the abundance and localization of CML, we developed an immunoelectron microscopic (IEM) detection method using anti-CML monoclonal antibody 6D12 in conjunction with computer-assisted image analysis. Antibody was pre-absorbed with purified AGE-bovine serum albumin to assure specificity. Antigen-antibody (ag-ab) complexes were individually identified with protein A-conjugated colloidal gold and counted with an automated system. We applied this method in 21 patients (pts) undergoing epicardial biopsy during coronary bypass grafting (CBG) [20 M, 1 F; mean age 65 +/- 7.4 (+/- SEM) years]. Seven pts had neither DM nor HTN, seven had HTN, and seven had DM + HTN. In contrast to the prior light microscopic studies, we detected CML scattered throughout the cardiomyocyte in all pts, but in widely varying amounts. Ag-ab complexes were abundant in sections through myofilaments (mean count 23.6 +/- 9.2 per microm(2), range 9.4-48) and even more so in mitochondria (mean count 34.4 +/- 11.9 per microm(2), range 14.1-68.2, P < 0.001 vs. myofilaments). CML was also detected in vascular endothelial cells. There were no statistically significant differences based on presence or absence of HTN or DM. In conclusion, our IEM method is the first to provide detailed delineation of the localization and abundance of CML in myocardium. CML is very prevalent in CBG pts, suggesting that AGEs could play a role in abnormal cardiomyocyte function, including altered energy metabolism.

Diabetes Mellitus Worsens Diastolic Left Ventricular Dysfunction in Aortic Stenosis Through Altered Myocardial Structure and Cardiomyocyte Stiffness

“…36 When light microscopic visualization of CML was implemented with electron microscopy, vascular deposition of CML was recently shown to be accompanied by interstitial accumulation of CML, which can directly reduce myocardial diastolic distensibility because of collagen crosslinking. 37 The findings of the present study differ from a previous study in HFREF and HFNEF patients free of coronary artery disease. 3 In the present study, DM worsened diastolic LV dysfunction in AS through fibrosis, AGE deposition, and raised cardiomyocyte F passive .…”

Myocardial Stiffness in Patients With Heart Failure and a Preserved Ejection Fraction

“…Intra-operative LV anterior wall epicardial biopsies were obtained as previously described (39, 40). Patients recruited to this protocol were part of an NIH grant (RO1HL089944) with multiple specific aims; therefore, biopsy samples were allocated to several protocols, data from some of which have been published 39, 40 . Given the size of each biopsy, all protocols could not be performed on each biopsy.…”

“…Anterior LV free wall epicardial biopsies weighing ∼25-50 mg were obtained during CABG soon after the patient was placed on cardiopulmonary bypass, as previously described 39,40 . The biopsy was placed in oxygenated HEPES-based Krebs buffer containing 30 mmol/L 2,3-butanedione monoximine (BDM) at room temperature 39, 40 .…”

Histochemistry and cell biology: the annual review 2010

“…Applying immunoelectron microscopy using an anti-carboxymethyl lysine (CML) antibody (CML is a major antigenic AGE structure) to localize and quantify CML in myocardial tissue obtained by epicardial biopsy during coronary bypass grafting (CBG), CML was found to be scattered throughout cardiomyocytes in all patients (independent of HTN or DM) and in endothelial cells. With CML being very prevalent in CBG patients, Donaldson et al (2010) concluded that AGEs could play a role in abnormal cardiomyocyte function. Franz et al (2010b) looked at chronic cardiac allograft rejection (CCAR), which is represented by cardiac allograft vasculopathy (CAV) and Wbrosis and zoomed into associated problems, i.e.…”

“…Abundance and distribution of advanced glycation endproducts (AGEs) were analysed by Donaldson et al (2010) in the human myocardium. AGE formation and accumulation are most accelerated under diabetes mellitus (DM), thus contributing to various complications (van Heerebeek et al 2008).…”